JP2010142234A

New glyphosate n-acetyltransferase (gat) gene

Abstract

Problem to be solved.To provide a method and reagent for making a living body (e.g. a plant) resistant to glyphosate.

Solution.This new glyphosate N-acetyltransferase (GAT) gene is an isolated polynucleotide or a recombinant polynucleotide, and contains the following: (a) a sequence selected from the group consisting of a plurality of specific amino acid sequences and nucleotide sequences encoding the amino acid sequences capable of being optimally aligned in an order by using a BLOSUM62 matrix, 11 gap existence penalties and 1 gap extension penalty so as to produce at least 430 similarity scores, or (b) complementary chain nucleotide sequences to the above.

Term

3.3 yearsto projected expiry

Projected expiry 29 December 2029, counted from filing; an application has no term until it is granted.

1 claim: 1 independent, 0 dependent

1
A gene as described herein. 本願明細書に記載されるような、遺伝子。

325 paragraphs, as filed

(Citation of related application) This application claims priority and interests in US Provisional Patent Application No. 60 / 244,385 filed October 30, 2000, the disclosure of which is hereby in its entirety for all purposes. It is used as a reference.

(Copyright notice in accordance with 37 CFR §1.71 (E)) Some of the disclosures in this patent document include material subject to copyright protection. The owner of this copyright does not refuse any reproduction of the Patent Documents and Published Patent Information in the US Patent and Trademark Office patent files or records, but in any other case. The copyright of is also protected.

(Background of invention) The crop selectivity for a specific herbicide can be imparted by the transfer of a gene encoding a suitable herbicide metabolizing enzyme into the crop. In some cases, these enzymes, and the nucleic acids that encode them, originate from plants. In other cases, these enzymes, and the nucleic acids that encode them, are derived from other organisms (eg, microorganisms). For example, Padgette et al. (1996) "New weed control oppotunities: Development of soybeans with a Round UP Ready"TM See gene, Herbicide-Resistant Crops (Duke), pages 54-84, CRC Press, Boca Raton; and Vasil (1996) "Phosphinothricin-resistant crops", Herbicide-Resistant Crops (Duke), pages 85-91. That thing. In fact, transgenic plants have been engineered to express different herbicide resistance / herbicide metabolism genes from different organisms. For example, acetohydroxy acid synthase, which has been found to make plants expressing this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants ( See, for example, Hattori et al. (1995) Mol Gen Genet 246: 419). Other genes that confer herbicide resistance: genes encoding chimeric proteins of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al. (1994) Plant Physiol Plant Physiol 106: 17), genes for glutathione reductase and superoxide dismutase (Aono et al. (1995) Plant Cell Physiol 36: 1687), and genes for various phosphotransferases (Datta et al. (1992) Plant Mol Biol 20: 619). Can be mentioned.

One herbicide that has been the subject of much research in this regard is N-phosphonomethylglycine (commonly referred to as glyphosate). Glyphosate is the world's best-selling herbicide, with sales estimated to reach $ 5 billion by 2003. Glyphosate is a broad spectrum herbicide that kills both hardwoods and lawn-type plants. A successful mode of commercial-level glyphosate resistance in transgenic plants is the modified Agrobacterium CP4. By introduction of the 5-enolpyruvyl shikimate-3-phosphate synthase (hereinafter referred to as EPSP synthase or EPSPS) gene. The transgene is directed to chloroplasts capable of continuing EPSP synthesis from phosphoenolpyruvate (PEP) and shikimic acid-3-phosphate in the presence of glyphosate. In contrast, native EPSP synthase is inhibited by glyphosate. Without the transgene, glyphosate-sprayed plants die rapidly due to inhibition of EPSP synthase, which blocks the downstream pathways required for the biosynthesis of aromatic amino acids, hormones, and vitamins. CP4 glyphosate-resistant soybean transgenic plants, for example by Monsanto, "Round UP Ready"TMIt is sold under the name.

In the environment, the predominant mechanism by which glyphosate is degraded is mediated by soil microbial flora metabolism. The major metabolite of glyphosate in soil was identified as aminomethyl phosphate (AMPA), which is ultimately converted to ammonia, phosphate and carbon dioxide. A proposed metabolic system that describes the degradation of glyphosate in soil via the AMPA pathway is shown in Figure 8. The sarcosine pathway, an alternative metabolic pathway for glyphosate-induced dysfunction of certain soil bacteria, occurs via the initial cleavage of the CP binding that yields inorganic phosphate and sarcosine, as illustrated in FIG.

Another successful herbicide / transgenic crop package is glufosinate (phosphinosricin) and Liberty Link.TMIt is a trait, which is commercially available, for example, by Aventis. Glufosinate is also a broad spectrum herbicide. The target of glufosinate is the chloroplast glutamate synthase enzyme. Resistant plants carry the bar gene from Streptomyces hygroscopicus and acquire resistance by the N-acetylation activity of the bar, which modifies and detoxifies glufosinate.

An enzyme capable of acetylating AMPA's primary amines is reported in PCT Application No. WO 00/29596. It was not stated that this enzyme could acetylate compounds with secondary amines (eg glyphosate).

Although various herbicide resistance strategies such as those described above may be available, further efforts have considerable commercial value. The present invention provides, for example, novel polynucleotides and polypeptides for conferring herbicide resistance, as well as a number of other advantages as will become apparent during the studies of the present disclosure.

The present invention provides:(1) An isolated polynucleotide or a recombinant polynucleotide, which is as follows:(a) Consists of SEQ ID NO: 300, SEQ ID NO: 445, and SEQ ID NO: 457 to generate a similarity score of at least 430 using the BLOSUM62 matrix, 11 gap extense penalties, and 1 gap extension penalty. A nucleotide sequence that encodes a sequence selected from the group and an amino acid sequence that can be optimally arranged; or(b) These complementary strand nucleotide sequences,Containing, polynucleotide.(2) The isolated polynucleotide or recombinant polynucleotide according to item 1, wherein the polypeptide has glyphosate N-acetyltransferase activity.(3) The polypeptide is at least 10 mM with respect to glyphosate.-1 min-12. The isolated or recombinant polynucleotide according to item 2, which catalyzes the acetylation of glyphosate at kcat / Km.(4) The isolated polynucleotide or recombinant polynucleotide according to item 2, wherein the polypeptide catalyzes the acetylation of aminomethylphosphonic acid.(5) An isolated or recombinant polynucleotide comprising a nucleotide sequence encoding a polypeptide having glyphosate N-acetyltransferase activity, wherein the polynucleotides are SEQ ID NO: 300, SEQ ID NO: 445 and SEQ ID NO: A polynucleotide comprising an amino acid sequence containing at least 20 contiguous amino acids of an amino acid sequence selected from the group consisting of 457.(6) The single according to item 5, wherein the polypeptide comprises an amino acid sequence containing at least 50 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO: 300, SEQ ID NO: 445 and SEQ ID NO: 457. Separated or recombinant polynucleotide.(7) The single according to item 5, wherein the polypeptide comprises an amino acid sequence containing at least 100 contiguous amino acids of the amino acid sequence selected from the group consisting of SEQ ID NO: 300, SEQ ID NO: 445 and SEQ ID NO: 457. Separated or recombinant polynucleotide.(8) The single according to item 5, wherein the polynucleotide contains an amino acid sequence containing about 140 consecutive amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO: 300, SEQ ID NO: 445 and SEQ ID NO: 457. Separated or recombinant polynucleotide.(9) The isolated polynucleotide or recombinant polynucleotide according to item 5, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 300, SEQ ID NO: 445 and SEQ ID NO: 457.(10) The isolated polynucleotide or recombinant polynucleotide according to item 5, which comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 193 and SEQ ID NO: 205.(11) The polynucleotide according to item 1, wherein the parent codon is replaced with a codon that is preferentially used in the plant with respect to the parent codon.(12) The polynucleotide according to item 1, further comprising a nucleotide sequence encoding an N-terminal chloroplast transport peptide.(13) A non-native variant of the polynucleotide according to item 1, wherein one or more amino acids of the encoded polypeptide have been mutated.(14) A nucleic acid construct containing the polynucleotide according to item 1.(15) The nucleic acid construct of item 14, comprising a promoter operably linked to the polynucleotide of item 1, wherein the promoter is heterologous to the polynucleotide and said nucleic acid. A nucleic acid construct that is effective in inducing expression of the encoded polypeptide sufficient to enhance glyphosate resistance in plant cells transformed with the construct.(16) The nucleic acid construct according to item 14, wherein the polynucleotide sequence according to item 1 functions as a selectable marker.(17) The nucleic acid construct according to item 14, wherein the construct is a vector.(18) Item 17, wherein the second polypeptide comprises a second polynucleotide sequence encoding the second polypeptide, which imparts detectable phenotypic properties to cells or tissues expressing at effective levels. Vector.(19) The vector according to item 18, wherein the detectable phenotypic characteristic functions as a selectable marker.(20) The vector according to item 19, wherein the detectable phenotypic property comprises a herbicide resistance, a pest resistance, or a visible marker.(21) The vector according to item 17, wherein the vector contains a T-DNA sequence.(22) The vector according to item 17, wherein the polynucleotide is operably linked to a regulatory sequence.(23) The vector according to item 17, wherein the vector is a plant transformation vector.(24) An isolated or recombinant polynucleotide, such as:(a) Substantially hybridizing nucleotides of a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 300, SEQ ID NO: 445 and SEQ ID NO: 457 under stringent conditions;(b) These complementary strand nucleotide sequences; or(c) Fragment of (a) or (b) encoding a polypeptide having glyphosate N-acetyltransferase activity,Containing, polynucleotide.(25) The polynucleotide according to item 24, which comprises a nucleotide sequence encoding glyphosate N-acetyltransferase.(26) A composition containing two or more polynucleotides according to item 1.(27) The composition according to item 26, which comprises at least 10 polynucleotides according to item 1.(28) A cell containing at least one polynucleotide according to item 1, wherein the polynucleotide is heterologous to the cell.(29) The cell of item 28, wherein the polynucleotide is operably linked to a regulatory sequence.(30) Cells transduced by the vector according to item 17.(31) The cell according to item 28 or item 30, wherein the cell is a transgenic plant cell.(32) The transgenic plant cell according to item 31, wherein the plant cell expresses a foreign polypeptide having glyphosate N-acetyltransferase activity.(33) A transgenic plant or transgenic plant explant containing the cells according to item 32.(34) The transgenic plant or transgenic plant explant according to item 33, wherein the plant or the plant explant expresses a polypeptide having glyphosate N-acetyltransferase activity.(35) The transgenic plant or plant explants are of the following genera: Eleusine, Lollium, Bambusa, Brassica, Dactylis, Sorghum, Pennisetum, Zea, Hordeum, Triticum, Secret, Avena, Hordeum, Saccharum, Coix, Glycine And the transgenic plant or transgenic plant explants according to item 34, which is a crop selected from among Gossypium.(36) The transgenic plant or transgenic plant explant according to item 34, wherein the transgenic plant or plant explant is Arabidosis.(37) The transgenic plant or transgenic plant explant according to item 34, wherein the transgenic plant or the transgenic plant explant is Gossypium.(38) The transgenic plant or trans of item 34, wherein the plant or plant explant exhibits enhanced resistance to glyphosate when compared to wild-type plants of the same genus, strain or variety. Transgenic plant explants.(39) Seeds produced by the plant according to item 34.(40) At least 10 mM for glyphosate-1 min-1A transgenic plant containing a heterologous gene encoding glyphosate N-acetyltransferase with kcat / Km, said plant lacking the heterologous gene without a significant reduction in yield due to the application of herbicides. A transgenic plant that is resistant to glyphosate applied at effective levels to inhibit the growth of the plant.(41) The transgenic plant according to item 40, wherein glyphosate N-acetyltransferase catalyzes the acetylation of aminomethylphosphonic acid.(42) A group consisting of SEQ ID NO: 300, SEQ ID NO: 445, and SEQ ID NO: 457 to generate a similarity score of at least 430 using the BLOSUM62 matrix, 11 gap existence penalties, and 1 gap extension penalty. An isolated polypeptide or recombinant polypeptide comprising a sequence selected from and an amino acid sequence that can be optimally aligned, wherein the polypeptide has glyphosate N-acetyltransferase activity.(43) The polypeptide is at least 10 mM with respect to glyphosate.-1 min-142. The isolated or recombinant polypeptide according to item 42, which catalyzes the acetylation of glyphosate at kcat / Km.(44) The polypeptide is at least 100 mM with respect to glyphosate.-1 min-143. The isolated or recombinant polypeptide according to item 43, which catalyzes the acetylation of glyphosate at kcat / Km.(45) The isolated or recombinant polypeptide of item 44, wherein the polypeptide catalyzes the acetylation of aminomethylphosphonic acid.(46) An isolated or recombinant polypeptide having glyphosate N-acetyltransferase activity, at least 20 contiguous amino acids in the amino acid sequence selected from the group consisting of SEQ ID NO: 445 and SEQ ID NO: 457. A polypeptide comprising an amino acid sequence comprising.(47) The isolate according to item 46, wherein the polypeptide comprises an amino acid sequence comprising at least 50 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO: 300, SEQ ID NO: 445 and SEQ ID NO: 457. Polypeptide or recombinant polypeptide.(48) The isolation according to item 46, wherein the polypeptide comprises an amino acid sequence comprising at least 100 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO: 300, SEQ ID NO: 445 and SEQ ID NO: 457. Polypeptide or recombinant polypeptide.(49) The isolation according to item 46, wherein the polypeptide comprises an amino acid sequence comprising approximately 140 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO: 300, SEQ ID NO: 445 and SEQ ID NO: 457. Polypeptide or recombinant polypeptide.(50) The isolated or recombinant polypeptide of item 46, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 300, SEQ ID NO: 445 and SEQ ID NO: 457.(51) The polynucleotide sequence of item 42, further comprising an N-terminal chloroplast transport peptide.(52) The non-native variant of the polypeptide according to claim 42, wherein one or more amino acids of the polypeptide have been mutated.(53) The non-native variant of the polypeptide of item 42, wherein one or more amino acids of the polypeptide have been modified relative to the parent polypeptide.(54) The polypeptide of item 53, wherein the polypeptide is produced by a variety of production procedures.(55) The polypeptide of item 54, wherein the various production procedures comprise a mutation or recombination of at least one parent polynucleotide encoding a glyphosate N-acetyltransferase polypeptide.(56) The polypeptide of item 55, wherein the parent polynucleotide is the polynucleotide of item 1.(57) The polypeptide of item 42, comprising a secretory or localized sequence.(58) The polypeptide of item 57, comprising a chloroplast transport sequence.(59) A polypeptide specifically bound by a polyclonal antiserum induced to one or more antigens, wherein the antigen is selected from the group consisting of SEQ ID NO: 300, SEQ ID NO: 445 and SEQ ID NO: 457. A polypeptide containing an amino acid sequence.(60) A polypeptide having GAT activity, which is as follows:(a) km to glyphosate, at least about 2 mM or less;(b) at least about 200 μM or less, km for acetyl-CoA; and(c) kcat, equal to at least about 6 / min,A polypeptide characterized by.(61) A method for producing glyphosate-resistant transgenic plants or glyphosate-resistant plant cells, which include:(a) The step of transforming a polynucleotide encoding glyphosate N-acetyltransferase into a plant or plant cell: and(b) A step of regenerating a transgenic plant from the transformed plant cells as needed,Including, methods.(62) The method according to item 61, wherein the polynucleotide is the polynucleotide according to item 1.(63) The method of item 61, wherein the polynucleotide is from a bacterial source.(64) Item 61, which comprises the step of growing transformed or transformed plant cells at a glyphosate concentration that inhibits the growth of wild-type plants of the same genus and does not inhibit the growth of transformed plants. The method described in.(65) The method of item 64, comprising growing the transformed plant or the transformed plant cell, or the progeny of the plant or the progeny of the plant cell in increasing concentrations of glyphosate.(66) The method of item 64, comprising the step of growing the transformed plant or the transformed plant cell at a glyphosate concentration that is lethal to the wild-type plant or wild-type plant cell of the same genus. ..(67) The method of item 62, comprising the step of growing a plant transformed with the polynucleotide of item 1.(68) The method of item 67, wherein the first plant is propagated by mating the first plant with the second plant, resulting in at least some of the offspring of the mating exhibiting glyphosate resistance.(69) A method for producing a variant of the polynucleotide according to item 1, wherein the polynucleotide according to item 1 is recursively recombined using a second polynucleotide, thereby producing a variant of the polynucleotide. A method comprising the step of forming a library.(70) The method of item 69, comprising selecting a variant polynucleotide from the library based on glyphosate N-acetyltransferase activity.(71) The method of item 70, wherein the recombination is performed in vitro.(72) The method of item 70, wherein the recombination is performed in vivo.(73) The method of item 70, wherein the recombination is performed in silico.(74) The method of item 70, wherein the recombination comprises family shuffling.(75) The method of item 70, wherein the recombination comprises a synthetic shuffling method.(76) The method of item 70, comprising replacing at least one parent codon in the nucleotide sequence with a codon that is preferentially used in the plant for that parent codon.(77) A library of modified polynucleotides produced by the method described in item 70.(78) A cell population comprising the library according to item 77.(79) A polypeptide produced by the method of item 70, wherein the recombinant polynucleotide encodes a polypeptide having glyphosate N-acetyltransferase activity.(80) A cell comprising the polynucleotide according to item 79.(81) The cell according to item 80, wherein the cell is a plant cell.(82) The cell according to item 81, wherein the cell is a transgenic plant cell.(83) Seeds produced by the plant according to item 82.(84) A polypeptide encoded by the polynucleotide according to item 79.(85) A method for producing a variant of the polynucleotide according to item 1, which comprises the step of mutating the polynucleotide.(86) A polynucleotide produced by the method according to item 85.(87) A method of selecting a plant or cell containing a nucleic acid construct, the following:(a) A step of providing a transgenic plant or transgenic cell containing a nucleic acid construct, wherein the nucleic acid construct comprises a nucleotide sequence encoding glyphosate N-acetyltrasferase;(b) The step of growing the plant or cells in the presence of glyphosate under conditions where the glyphosate N-acetyltransferase is expressed at effective levels, thereby the transgenic plant or the transgenic. A step in which a cell grows at a significantly faster rate than if the plant or the cell did not contain the nucleic acid construct.Including, methods.(88) The method of item 87, wherein the nucleic acid construct comprises the second nucleotide sequence, which encodes a polypeptide and regulatory sequence operably linked to the second nucleotide sequence.(89) A method of selectively controlling weeds in areas with crops, such as:(a) The step of planting glyphosate-tolerant crop seeds or crops in the region as a result of transformation with a gene encoding glyphosate N-acetyltransferase; and(b) The step of applying a sufficient amount of glyphosate to the crop and the weed in the area without significantly affecting the crop.Including, methods.(90) A method of producing a genetically transformed plant that is resistant to glyphosate:(a) Recombination, a step of inserting a double-stranded DNA molecule into the genome of a plant cell, wherein the DNA molecule is as follows: (i) Promoters that function in plant cells to generate RNA sequences (ii) A structural DNA sequence that produces an RNA sequence encoding the polypeptide according to item 42. (iii) Containing a 3'untranslated region that functions in plant cells to add a polyadenyl nucleotide stretch to the 3'end of the RNA sequence, the promoter is heterologous to each of the structural DNA sequences, and the DNA. A step adapted to cause sufficient expression of the encoded polypeptide to enhance the glyphosate resistance of the molecularly transformed plant cell.(b) Steps to acquire transformed plant cells; and(c) A step of regenerating a genetically transformed plant having increased glyphosate resistance from the transformed plant cell.Including, methods.(91) A method of producing crops, such as:(a) The step of growing a crop plant that is glyphosate resistant as a result of transformation of the gene encoding glyphosate N-acetyltransferase under conditions where the crop plant produces a crop; and(b) Step of collecting crops from the crop plantIncluding, methods.(92) The method of item 91, comprising applying glyphosate to the crop at a concentration effective for controlling weeds.(93) The method of item 92, wherein the crop is cotton, corn or soybean.(94) The isolated or recombinant polynucleotide according to item 1, wherein at least 90% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions:(a) 2, 4, 15, 19, 26, 28, 31, 45, 51, 54, 86, 90, 91, 97, 103, 105, 106, 114, 123, 129, 139 andAnd / or the amino acid residue at position 145 is B1; and(b) 3, 5, 8, 10, 11, 14, 17, 18, 24, 27, 32, 37, 38, 47, 48, 49, 52, 57, 58, 61, 62, 63, 68, 69 , 79, 80, 82, 83, 89, 92, 100, 101, 104, 119, 120, 124, 125, 126, 128, 131, 143 and / or the amino acid residue at position 144 is B2;Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y and V, and B2 is R, N, D, C, Q, E, G, An amino acid selected from the group consisting of H, K, P, S and T,Polynucleotide.(95) The isolated or recombinant polynucleotide according to item 1, wherein at least 80% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions:(a) The amino acid residue at position 2, 4, 15, 19, 26, 28, 51, 54, 86, 90, 91, 97, 103, 105, 106, 114, 129, 139 and / or 145 is Z1. is there;(b) The amino acid residue at position 31 and / or 45 is Z2;(c) The amino acid residue at position 8 and / or 89 is Z3;(d) The amino acid residue at positions 82, 92, 101 and / or 120 is Z4;(e) The amino acid residue at positions 3, 11, 27 and / or 79 is Z5;(f) The amino acid residue at position 123 is Z1 or Z2;(g) The amino acid residues at positions 12, 33, 35, 39, 53, 59, 112, 132, 135, 140 and / or 146 are Z1 or Z3;(h) The amino acid residue at position 30 is Z1 or Z4;(i) The amino acid residue at position 6 is Z1 or Z6;(j) The amino acid residue at position 81 and / or 113 is Z2 or Z3;(k) The amino acid residue at position 138 and / or 142 is Z2 or Z4;(l) The amino acid residues at positions 5, 17, 24, 57, 61, 124 and / or 126 are Z3 or Z4;(m) The amino acid residue at position 104 is Z3 or Z5;(o) The amino acid residues at positions 38, 52, 62 and / or 69 are Z3 or Z6;(p) The amino acid residues at positions 14, 119 and / or 144 are Z4 or Z5;(q) The amino acid residue at position 18 is Z4 or Z6;(r) The amino acid residues at positions 10, 32, 48, 63, 80 and / or 83 are Z5 or Z6;(s) The amino acid residue at position 40 is Z1, Z2 or Z3;(t) The amino acid residues at positions 65 and / or 96 are Z1, Z3 or Z5;(u) The amino acid residue at position 84 and / or 115 is Z1, Z3 or Z4;(v) The amino acid residue at position 93 is Z2, Z3 or Z4;(w) The amino acid residue at position 130 is Z2, Z4 or Z6;(x) The amino acid residues at positions 47 and / or 58 are Z3, Z4 and Z6;(y) The amino acid residues at positions 49, 68, 100 and / or 143 are Z3, Z4 and Z5;(z) The amino acid residue at position 131 is Z3, Z5 or Z6;(aa) The amino acid residue at position 125 and / or 128 is Z4, Z5 or Z6;(ab) The amino acid residue at position 67 is Z1, Z3, Z4 or Z5;(ac) The amino acid residue at position 60 is Z1, Z4, Z5 or Z6;(ad) The amino acid residue at position 37 is Z3, Z4, Z5 or Z6;Where Z1 is an amino acid sequence selected from the group consisting of A, I, L, M and V; Z2 is an amino acid selected from the group consisting of F, W and Y; Z3 is N, Q, Amino acids selected from the group consisting of S, and T; Z4 is an amino acid selected from the group consisting of R, H and K; Z5 is an amino acid selected from the group consisting of D and E; Z6 is an amino acid selected from the group consisting of C, G and P,Polynucleotide.(96) The isolated or recombinant polynucleotide according to item 1, wherein at least 90% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions:(a) 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121 and / Or the amino acid residue at position 141 is B1;(b) 16, 21, 22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108, 109 , 111, 116, 122, 127, 133, 134, 136 and / or the amino acid residue at position 137 is B2;Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y and V, and B2 is R, N, D, C, Q, E, G, H. , An amino acid selected from the group consisting of K, P, S and T,Polynucleotide.(97) The isolated or recombinant polynucleotide according to item 1, wherein at least 90% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) 1st, 7th, 9th, 20th, 36th, 42nd, 50th, 64th, 72nd, 75th, 76th, 78th, 94th, 98th, 110th, 121st And / or the amino acid residue at position 141 is Z1;(b) The amino acid residues at positions 13, 46, 56, 70, 107, 117 and / or 118 are Z2;(c) The amino acid residues at positions 23, 55, 71, 77, 88 and / or 109 are Z3;(d) The amino acid residues at positions 16, 21, 41, 73, 85, 99 and / or 111 are Z4;(e) The amino acid residue at positions 34 and / or 95 is Z5;(f) 22nd, 25th, 29th, 43rd, 44th, 66th, 74th, 87th, 102nd, 108th, 116th, 122nd, 127th, 133th, 134th, 136th And / or the amino acid residue at position 137 is Z6; Where Z1 is an amino acid selected from the group consisting of A, I, L, M, and V; Z2 is an amino acid selected from the group consisting of F, W, and Y; Z3. Is an amino acid selected from the group consisting of N, Q, S, and T; Z4 is an amino acid selected from the group consisting of R, H, and K; Z5 is selected from the group consisting of D and E. It is an amino acid selected from the group consisting of; and Z6 is an amino acid selected from the group consisting of C, G, and P.(98) The isolated or recombinant polynucleotide according to item 94, wherein at least 90% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) 1st, 7th, 9th, 13th, 20th, 36th, 42nd, 46th, 50th, 56th, 64th, 70th, 72nd, 75th, 76th, 78th , 94, 98, 107, 110, 117, 118, 121 and / or 141 amino acid residues are B1; and(b) 16th, 21st, 22nd, 23rd, 25th, 29th, 34th, 41st, 43rd, 44th, 55th, 66th, 71st, 73rd, 74th, 77th , 85th, 87th, 88th, 95th, 99th, 102nd, 108th, 109th, 111th, 116th, 122nd, 127th, 133th, 134th, 136th and / or 137th The amino acid residue of is B2; Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y and V; and B2 is R, N, D, C, Q, E, An amino acid selected from the group consisting of G, H, K, P, S, and T.(99) The isolated or recombinant polynucleotide according to item 94, wherein at least 90% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) 1st, 7th, 9th, 13th, 20th, 36th, 42nd, 46th, 50th, 56th, 64th, 70th, 72nd, 75th, 76th, 78th , 94, 98, 107, 110, 117, 118, 121 and / or 141 amino acid residues are B1; and(b) 16th, 21st, 22nd, 23rd, 25th, 29th, 34th, 41st, 43rd, 44th, 55th, 66th, 71st, 73rd, 74th, 77th , 85th, 87th, 88th, 95th, 99th, 102nd, 108th, 109th, 111th, 116th, 122nd, 127th, 133th, 134th, 136th and / or 137th The amino acid residue of is B2; Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y and V; and B2 is R, N, D, C, Q, E, An amino acid selected from the group consisting of G, H, K, P, S, and T.(100) The isolated or recombinant polynucleotide according to item 94, wherein at least 90% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) 1st, 7th, 9th, 13th, 20th, 36th, 42nd, 46th, 50th, 56th, 64th, 70th, 72nd, 75th, 76th, 78th , 94, 98, 107, 110, 117, 118, 121 and / or 141 amino acid residues are B1; and(b) 16th, 21st, 22nd, 23rd, 25th, 29th, 34th, 41st, 43rd, 44th, 55th, 66th, 71st, 73rd, 74th, 77th , 85th, 87th, 88th, 95th, 99th, 102nd, 108th, 109th, 111th, 116th, 122nd, 127th, 133th, 134th, 136th and / or 137th The amino acid residue of is B2; Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y and V; and B2 is R, N, D, C, Q, E, An amino acid selected from the group consisting of G, H, K, P, S, and T.(101) The isolated or recombinant polynucleotide according to item 95, wherein at least 90% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) 1st, 7th, 9th, 13th, 20th, 36th, 42nd, 46th, 50th, 56th, 64th, 70th, 72nd, 75th, 76th, 78th , 94, 98, 107, 110, 117, 118, 121 and / or 141 amino acid residues are B1; and(b) 16th, 21st, 22nd, 23rd, 25th, 29th, 34th, 41st, 43rd, 44th, 55th, 66th, 71st, 73rd, 74th, 77th , 85th, 87th, 88th, 95th, 99th, 102nd, 108th, 109th, 111th, 116th, 122nd, 127th, 133th, 134th, 136th and / or 137th The amino acid residue of is B2; Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y and V; and B2 is R, N, D, C, Q, E, An amino acid selected from the group consisting of G, H, K, P, S, and T.(102) An isolated or recombinant polynucleotide according to item, wherein at least 80% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations: nucleotide:(a) The amino acid residue at position 2 is I or L;(b) The amino acid residue at position 3 is E or D;(c) The amino acid residue at position 4 is V, A or I;(d) The amino acid residue at position 5 is K, R or N;(e) The amino acid residue at position 6 is P or L;(f) The amino acid residue at position 8 is N, S or T;(g) The amino acid residue at position 10 is E or G;(h) The amino acid residue at position 11 is D or E;(i) The amino acid residue at position 12 is T or A;(j) The amino acid residue at position 14 is E or K;(k) The amino acid residue at position 15 is I or L;(l) The amino acid residue at position 17 is H or Q;(m) The amino acid residue at position 18 is R, C or K;(n) The amino acid residue at position 19 is I or V;(o) The amino acid residue at position 24 is Q or R;(p) The amino acid residue at position 26 is L or I;(q) The amino acid residue at position 27 is E or D;(r) The amino acid residue at position 28 is A or V;(s) The amino acid residue at position 30 is K, M or R;(t) The amino acid residue at position 31 is Y or F;(u) The amino acid residue at position 32 is E or G;(v) The amino acid residue at position 33 is T, A or S;(w) The amino acid residue at position 35 is L, S or M;(x) The amino acid residue at position 37 is R, G, E or Q;(y) The amino acid residue at position 38 is G or S;(z) The amino acid residue at position 39 is T, A or S;(aa) The amino acid residue at position 40 is F, L or S;(ab) The amino acid residue at position 45 is Y or F;(ac) The amino acid residue at position 47 is R, Q or G;(ad) The amino acid residue at position 48 is G or D;(ae) The amino acid residue at position 49 is K, R, E or Q;(af) The amino acid residue at position 51 is I or V;(ag) The amino acid residue at position 52 is S, C or G;The amino acid residue at position (ah) 53 is I or T;(ai) The amino acid residue at position 54 is A or V;(aj) The amino acid residue at position 57 is H or N;The amino acid residue at position (ak) 58 is Q, K, N or P;The amino acid residue at position (al) 59 is A or S;(am) The amino acid residue at position 60 is E, K, G, V or D;The amino acid residue at position (an) 61 is H or Q;The amino acid residue at position (ao) 62 is P, S or T;(ap) The amino acid residue at position 63 is E, G or D;(aq) The amino acid residue at position 65 is E, D, V or Q;The amino acid residue at position (ar) 67 is Q, E, R, L, H or K;(as) The amino acid residue at position 68 is K, R, E or N;(at) The amino acid residue at position 69 is Q or P;(au) The amino acid residue at position 79 is E or D;(av) The amino acid residue at position 80 is G or E;(aw) The amino acid residue at position 81 is Y, N or F;The amino acid residue at position (ax) 82 is R or H;The amino acid residue at position (ay) 83 is E, G or D;The amino acid residue at position (az) 84 is Q, R or L;(ba) The amino acid residue at position 86 is A or V;The amino acid residue at position (bb) 89 is T or S;(bc) The amino acid residue at position 90 is L or I;(bd) The amino acid residue at position 91 is I or V;(be) The amino acid residue at position 92 is R or K;The amino acid residue at position (bf) 93 is H, Y or Q;The amino acid residue at position (bg) 96 is E, A or Q;The amino acid residue at position (bh) 97 is L or I;(bi) The amino acid residue at position 100 is K, R, N or E;(bj) The amino acid residue at position 101 is K or R;The amino acid residue at position (bk) 103 is A or V;(bl) The amino acid residue at position 104 is D or N;The amino acid residue at position (bm) 105 is L or M;The amino acid residue at position (bn) 106 is L or I;(bo) The amino acid residue at position 112 is T or I;The amino acid residue at position (bp) 113 is S, T or F;The amino acid residue at position (bq) 114 is A or V;(br) The amino acid residue at position 115 is S, R or A;(bs) The amino acid residue at position 119 is K, E or R;(bt) The amino acid residue at position 120 is K or R;(bu) The amino acid residue at position 123 is F or L;The amino acid residue at position (bv) 124 is S or R;The amino acid residue at position (bw) 125 is E, K, G or D;The amino acid residue at position (bx) 126 is Q or H;The amino acid residue at position (by) 128 is E, G or K;The amino acid residue at position (bz) 129 is V, I or A;(ca) The amino acid residue at position 130 is Y, H, F or C;(cb) The amino acid residue at position 131 is D, G, N or E;The amino acid residue at position (cc) 132 is I, T, A, M, V or L;The amino acid residue at position (cd) 135 is V, T, A or I;(ce) The amino acid residue at position 138 is H or Y;(cf) The amino acid residue at position 139 is I or V;The amino acid residue at position (cg) 140 is L or S;The amino acid residue at position (ch) 142 is Y or H;(ci) The amino acid residue at position 143 is K, T or E;The amino acid residue at position (cj) 144 is K, E or R;The amino acid residue at position (ck) 145 is L or I; andThe amino acid residue at position (cl) 146 is T or A.(103) The isolated or recombinant polynucleotide according to item 1, wherein at least 80% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) The amino acid residues at positions 9, 76, 94 and 110 are A;(b) The amino acid residues at positions 29 and 108 are C;(c) The amino acid residue at position 34 is D;(d) The amino acid residue at position 95 is E;(e) The amino acid residue at position 56 is F;(f) The amino acid residues at positions 43, 44, 66, 74, 87, 102, 116, 122, 127 and 136 are G;(g) The amino acid residue at position 41 is H;(h) The amino acid residue at position 7 is I;(i) The amino acid residue at position 85 is K;(j) The amino acid residues at positions 20, 36, 42, 50, 72, 78, 98 and 121 are L;(k) The amino acid residues at positions 1, 75 and 141 are M;(l) The amino acid residues at positions 23, 64 and 109 are N;(m) The amino acid residues at positions 22, 25, 133, 134 and 137 are P;(n) The amino acid residue at position 71 is Q;(o) The amino acid residues at positions 16, 21, 73, 99 and 111 are R;(p) The amino acid residues at positions 55 and 88 are S;(q) The amino acid residue at position 77 is T;(r) The amino acid residue at position 107 is W; and(s) The amino acid residues at positions 13, 46, 70, 117 and 118 are Y.(104) The isolated or recombinant polynucleotide according to item 102, wherein at least 90% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) 1st, 7th, 9th, 13th, 20th, 36th, 42nd, 46th, 50th, 56th, 64th, 70th, 72nd, 75th, 76th, 78th , 94, 98, 107, 110, 117, 118, 121 and / or 141 amino acid residues are B1; and(b) 16th, 21st, 22nd, 23rd, 25th, 29th, 34th, 41st, 43rd, 44th, 55th, 66th, 71st, 73rd, 74th, 77th , 85th, 87th, 88th, 95th, 99th, 102nd, 108th, 109th, 111th, 116th, 122nd, 127th, 133th, 134th, 136th and / or 137th The amino acid residue of is B2; Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y and V; and B2 is R, N, D, C, Q, E, An amino acid selected from the group consisting of G, H, K, P, S, and T.(105) The isolated or recombinant polynucleotide according to item 103, wherein at least 90% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) 2nd, 4th, 15th, 19th, 26th, 28th, 31st, 45th, 51st, 54th, 86th, 90th, 91st, 97th, 103rd, 105th , 106, 114, 123, 129, 139 and / or 145 amino acid residues are B1;(b) 3rd, 5th, 8th, 10th, 11th, 14th, 17th, 18th, 24th, 27th, 32nd, 37th, 38th, 47th, 48th, 49th , 52nd, 57th, 58th, 61st, 62nd, 63rd, 68th, 69th, 79th, 80th, 82nd, 83rd, 89th, 92nd, 100th, 101st, 104th The amino acid residues at positions 119, 120, 124, 125, 126, 128, 131, 143 and / or 144 are B2; Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y and V; and B2 is R, N, D, C, Q, E, An amino acid selected from the group consisting of G, H, K, P, S, and T.(106) The isolated or recombinant polynucleotide according to item 102, wherein at least 90% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) 1st, 7th, 9th, 20th, 36th, 42nd, 50th, 64th, 72nd, 75th, 76th, 78th, 94th, 98th, 110th, 121st And / or the amino acid residue at position 141 is Z1;(b) The amino acid residues at positions 13, 46, 56, 70, 107, 117 and / or 118 are Z2;(c) The amino acid residues at positions 23, 55, 71, 77, 88 and / or 109 are Z3;(d) The amino acid residues at positions 16, 21, 41, 73, 85, 99 and / or 111 are Z4;(e) The amino acid residue at positions 34 and / or 95 is Z5;(f) 22nd, 25th, 29th, 43rd, 44th, 66th, 74th, 87th, 102nd, 108th, 116th, 122nd, 127th, 133th, 134th, 136th And / or the amino acid residue at position 137 is Z6; Where Z1 is an amino acid selected from the group consisting of A, I, L, M, and V; Z2 is an amino acid selected from the group consisting of F, W, and Y; Z3. Is an amino acid selected from the group consisting of N, Q, S, and T; Z4 is an amino acid selected from the group consisting of R, H, and K; Z5 is selected from the group consisting of D and E. It is an amino acid selected from the group consisting of; and Z6 is an amino acid selected from the group consisting of C, G, and P.(107) The isolated or recombinant polynucleotide according to item 103, wherein at least 80% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) 2nd, 4th, 15th, 19th, 26th, 28th, 51st, 54th, 86th, 90th, 91st, 97th, 103rd, 105th, 106th, 114th , 129, 139 and / or 145 amino acid residues are Z1;(b) The amino acid residue at positions 31 and / or 45 is Z2;(c) The amino acid residue at positions 8 and / or 89 is Z3;(d) The amino acid residue at positions 82, 92, 101 and / or 120 is Z4;(e) The amino acid residue at positions 3, 11, 27 and / or 79 is Z5;(f) The amino acid residue at position 123 is Z1 or Z2;(g) The amino acid residues at positions 12, 33, 35, 39, 53, 59, 112, 132, 135, 140 and / or 146 are Z1 or Z3;(h) The amino acid residue at position 30 is Z1 or Z4;(i) The amino acid residue at position 6 is Z1 or Z6;(j) The amino acid residues at positions 81 and / or 113 are Z2 or Z3;(k) The amino acid residues at positions 138 and / or 142 are Z2 or Z4;(l) The amino acid residues at positions 5, 17, 24, 57, 61, 124 and / or 126 are Z3 or Z4;(m) The amino acid residue at position 104 is Z3 or Z5;(o) The amino acid residues at positions 38, 52, 62 and / or 69 are Z3 or Z6;(p) The amino acid residues at positions 14, 119 and / or 144 are Z4 or Z5;(q) The amino acid residue at position 18 is Z4 or Z6;(r) The amino acid residues at positions 10, 32, 48, 63, 80 and / or 83 are Z5 or Z6;(s) The amino acid residue at position 40 is Z1, Z2 or Z3;(t) The amino acid residues at positions 65 and / or 96 are Z1, Z3 or Z5;(u) The amino acid residues at positions 84 and / or 115 are Z1, Z3 or Z4;(v) The amino acid residue at position 93 is Z2, Z3 or Z4;(w) The amino acid residue at position 130 is Z2, Z4 or Z6;(x) The amino acid residues at positions 47 and / or 58 are Z3, Z4 or Z6;(y) The amino acid residues at positions 49, 68, 100 and / or 143 are Z3, Z4 or Z5;(z) The amino acid residue at position 131 is Z3, Z5 or Z6;(aa) The amino acid residues at positions 125 and / or 128 are Z4, Z5 or Z6;(ab) The amino acid residue at position 67 is Z1, Z3, Z4 or Z5;(ac) The amino acid residue at position 60 is Z1, Z4, Z5 or Z6; and(ad) The amino acid residue at position 37 is Z3, Z4, Z5 or Z6; Where Z1 is an amino acid selected from the group consisting of A, I, L, M, and V; Z2 is an amino acid selected from the group consisting of F, W, and Y; Z3. Is an amino acid selected from the group consisting of N, Q, S, and T; Z4 is an amino acid selected from the group consisting of R, H, and K; Z5 is selected from the group consisting of D and E. It is an amino acid selected from the group consisting of; and Z6 is an amino acid selected from the group consisting of C, G, and P.(108) The isolated or recombinant polynucleotide according to item 102, wherein at least 80% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) The amino acid residues at positions 9, 76, 94 and 110 are A;(b) The amino acid residues at positions 29 and 108 are C;(c) The amino acid residue at position 34 is D;(d) The amino acid residue at position 95 is E;(e) The amino acid residue at position 56 is F;(f) The amino acid residues at positions 43, 44, 66, 74, 87, 102, 116, 122, 127 and 136 are G;(g) The amino acid residue at position 41 is H;(h) The amino acid residue at position 7 is I;(i) The amino acid residue at position 85 is K;(j) The amino acid residues at positions 20, 36, 42, 50, 72, 78, 98 and 121 are L;(k) The amino acid residues at positions 1, 75 and 141 are M;(l) The amino acid residues at positions 23, 64 and 109 are N;(m) The amino acid residues at positions 22, 25, 133, 134 and 137 are P;(n) The amino acid residue at position 71 is Q;(o) The amino acid residues at positions 16, 21, 73, 99 and 111 are R;(p) The amino acid residues at positions 55 and 88 are S;(q) The amino acid residue at position 77 is T;(r) The amino acid residue at position 107 is W; and(s) The amino acid residues at positions 13, 46, 70, 117 and 118 are Y.(109) The isolated polynucleotide or recombinant polynucleotide according to item 1, wherein the amino acid residue corresponding to position 28 in the amino acid sequence is V.(110) The isolated or recombinant polynucleotide according to item 1, wherein the amino acid sequence is selected from the group consisting of SEQ ID NOs: 6-10 and 263-514. nucleotide.(111) The isolated or recombinant polynucleotide according to item 42, wherein at least 90% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) 2nd, 4th, 15th, 19th, 26th, 28th, 31st, 45th, 51st, 54th, 86th, 90th, 91st, 97th, 103rd, 105th , 106, 114, 123, 129, 139 and / or 145 amino acid residues are B1; and(b) 3rd, 5th, 8th, 10th, 11th, 14th, 17th, 18th, 24th, 27th, 32nd, 37th, 38th, 47th, 48th, 49th , 52nd, 57th, 58th, 61st, 62nd, 63rd, 68th, 69th, 79th, 80th, 82nd, 83rd, 89th, 92nd, 100th, 101st, 104th The amino acid residues at positions 119, 120, 124, 125, 126, 128, 131, 143 and / or 144 are B2; Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y and V; and B2 is R, N, D, C, Q, E, An amino acid selected from the group consisting of G, H, K, P, S, and T.(112) The isolated or recombinant polynucleotide according to item 42, wherein at least 80% of the amino acid residues corresponding to the following positions in the amino acid sequence are subject to the following limitations. Polynucleotide:(a) 2nd, 4th, 15th, 19th, 26th, 28th, 51st, 54th, 86th, 90th, 91st, 97th, 103rd, 105th, 106th, 114th , 129, 139 and / or 145 amino acid residues are Z1;(b) The amino acid residue at positions 31 and / or 45 is Z2;(c) The amino acid residue at positions 8 and / or 89 is Z3;(d) The amino acid residues at positions 82, 92, 101 and 120 are Z4;(e) The amino acid residue at positions 3, 11, 27 and / or 79 is Z5;(f) The amino acid residue at position 123 is Z1 or Z2;(g) The amino acid residues at positions 12, 33, 35, 39, 53, 59, 112, 132, 135, 140 and / or 146 are Z1 or Z3;(h) The amino acid residue at position 30 is Z1 or Z4;(i) The amino acid residue at position 6 is Z1 or Z6;(j) The amino acid residues at positions 81 and / or 113 are Z2 or Z3;(k) The amino acid residues at positions 138 and / or 142 are Z2 or Z4;(l) The amino acid residues at positions 5, 17, 24, 57, 61, 124 and / or 126 are Z3 or Z4;(m) The amino acid residue at position 104 is Z3 or Z5;(o) The amino acid residues at positions 38, 52, 62 and / or 69 are Z3 or Z6;(p) The amino acid residues at positions 14, 119 and / or 144 are Z4 or Z5;(q) The amino acid residue at position 18 is Z4 or Z6;(r) The amino acid residues at positions 10, 32, 48, 63, 80 and / or 83 are Z5 or Z6;(s) The amino acid residue at position 40 is Z1, Z2 or Z3;(t) The amino acid residues at positions 65 and / or 96 are Z1, Z3 or Z5;(u) The amino acid residues at positions 84 and / or 115 are Z1, Z3 or Z4;(v) The amino acid residue at position 93 is Z2, Z3 or Z4;(w) The amino acid residue at position 130 is Z2, Z4 or Z6;(x) The amino acid residues at positions 47 and / or 58 are Z3, Z4 or Z6;(y) The amino acid residues at positions 49, 68, 100 and / or 143 are Z3, Z4 or Z5;(z) The amino acid residue at position 131 is Z3, Z5 or Z6;(aa) The amino acid residues at positions 125 and / or 128 are Z4, Z5 or Z6;(ab) The amino acid residue at position 67 is Z1, Z3, Z4 or Z5;(ac) The amino acid residue at position 60 is Z1, Z4, Z5 or Z6; and(ad) The amino acid residue at position 37 is Z3, Z4, Z5 or Z6; Where Z1 is an amino acid selected from the group consisting of A, I, L, M, and V; Z2 is an amino acid selected from the group consisting of F, W, and Y; Z3. Is an amino acid selected from the group consisting of N, Q, S, and T; Z4 is an amino acid selected from the group consisting of R, H, and K; Z5 is selected from the group consisting of D and E. It is an amino acid selected from the group consisting of; and Z6 is an amino acid selected from the group consisting of C, G, and P.(113) At least 90% of the amino acid residues in the amino acid sequence corresponding to the following positions in the isolated or recombinant polypeptide of item 42 meet the following restrictions: (a) 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121 and / Or the amino acid residue at position 141 is B1; and (b) 16, 21, 22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108, 109 , 111, 116, 122, 127, 133, 134, 136 and / or the amino acid residue at position 137 is B2;Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y, and V; and B2 is R, N, D, C, Q, E, G, H, An amino acid selected from the group consisting of K, P, S and T,Polypeptide.(114) The isolated or recombinant polypeptide of item 42, wherein at least 90% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions: (a) The amino acid residue at positions 1, 7, 9, 20, 36, 42, 50, 64, 72, 75, 76, 78, 94, 98, 110, 121 and / or 141 is Z1; (b) The amino acid residue at positions 13, 46, 56, 70, 107, 117 and / or 118 is Z2; (c) The amino acid residue at positions 23, 55, 71, 77, 88 and / or 109 is Z3; (d) The amino acid residue at positions 16, 21, 41, 73, 85, 99 and / or 111 is Z4; (e) The amino acid residue at position 34 and / or 95 is Z5; (f) The amino acid residue at positions 22, 25, 29, 43, 44, 66, 74, 87, 102, 108, 116, 122, 127, 133, 134, 136 and / or 137 is Z6;Where Z1 is an amino acid selected from the group consisting of A, I, L, M and V; Z2 is an amino acid selected from the group consisting of F, W and Y; Z3 is an amino acid selected from the group consisting of N, Q, S and Z4 is an amino acid selected from the group consisting of T; Z4 is an amino acid selected from the group consisting of R, H and K; Z5 is an amino acid selected from the group consisting of D and E; and Z6 is an amino acid selected from the group consisting of C. , An amino acid selected from the group consisting of G and P,Polypeptide.(115) The isolated or recombinant polypeptide of item 111, wherein at least 90% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions: (a) 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121 and / Or the amino acid residue at position 141 is B1; (b) 16, 21, 22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108, 109 , 111, 116, 122, 127, 133, 134, 136 and / or the amino acid residue at position 137 is B2;Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y, and V; and B2 is R, N, D, C, Q, E, G, H, An amino acid selected from the group consisting of K, P, S and T,Polypeptide.(116) The isolated or recombinant polypeptide of item 111, wherein at least 90% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions: (a) 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121 and / Or the amino acid residue at position 141 is B1; (b) 16, 21, 22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108, 109 , 111, 116, 122, 127, 133, 134, 136 and / or the amino acid residue at position 137 is B2;Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y, and V; and B2 is R, N, D, C, Q, E, G, H, An amino acid selected from the group consisting of K, P, S and T,Polypeptide.(117) The isolated or recombinant polypeptide of item 111, wherein at least 90% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions: (a) 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121 and / Or the amino acid residue at position 141 is B1; (b) 16, 21, 22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108, 109 , 111, 116, 122, 127, 133, 134, 136 and / or the amino acid residue at position 137 is B2;Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y, and V; and B2 is R, N, D, C, Q, E, G, H, An amino acid selected from the group consisting of K, P, S and T,Polypeptide.(118) The isolated or recombinant polypeptide of item 112, wherein at least 90% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions: (a) 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121 and / Or the amino acid residue at position 141 is B1; (b) 16, 21, 22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108, 109 , 111, 116, 122, 127, 133, 134, 136 and / or the amino acid residue at position 137 is B2;Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y, and V; and B2 is R, N, D, C, Q, E, G, H, An amino acid selected from the group consisting of K, P, S and T,Polypeptide.(119) The isolated or recombinant polypeptide of item 42, wherein at least 80% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions: (a) The amino acid residue at position 2 is I or L; (b) The amino acid residue at position 3 is E or D; (c) The amino acid residue at position 4 is V, A or I; (d) The amino acid residue at position 5 is K, R or N; (e) The amino acid residue at position 6 is P or L; (f) The amino acid residue at position 8 is N, S or T; (g) The amino acid residue at position 10 is E or G; (h) The amino acid residue at position 11 is D or E; (i) The amino acid residue at position 12 is T or A; (j) The amino acid residue at position 14 is E or K; (k) The amino acid residue at position 15 is I or L; (l) The amino acid residue at position 17 is H or Q; (m) The amino acid residue at position 18 is R, C or K; (n) The amino acid residue at position 19 is I or V; (o) The amino acid residue at position 24 is Q or R; (p) The amino acid residue at position 26 is L or I; (q) The amino acid residue at position 27 is E or D; (r) The amino acid residue at position 28 is A or V; (s) The amino acid residue at position 30 is K, M or R; (t) The amino acid residue at position 31 is Y or F; (u) The amino acid residue at position 32 is E or G; (v) The amino acid residue at position 33 is T, A or S; (w) The amino acid residue at position 35 is L, S or M; (x) The amino acid residue at position 37 is R, G, E or Q; (y) The amino acid residue at position 38 is G or S; (z) The amino acid residue at position 39 is T, A or S; (aa) The amino acid residue at position 40 is F, L or S; (ab) The amino acid residue at position 45 is Y or F; (ac) The amino acid residue at position 47 is R, Q or G; (ad) The amino acid residue at position 48 is G or D; (ae) The amino acid residue at position 49 is K, R, E or Q; (af) The amino acid residue at position 51 is I or V; (ag) The amino acid residue at position 52 is S, C or G; (ah) The amino acid residue at position 53 is I or T; (ai) The amino acid residue at position 54 is A or V; (aj) The amino acid residue at position 57 is H or N; The amino acid residue at position (ak) 58 is Q, K, N or P; (al) The amino acid residue at position 59 is A or S; (am) The amino acid residue at position 60 is E, K, G, V or D; (an) The amino acid residue at position 61 is H or Q; (ao) The amino acid residue at position 62 is P, S or T; (ap) The amino acid residue at position 63 is E, G or D; (aq) The amino acid residue at position 65 is E, D, V or Q; The amino acid residue at position (ar) 67 is Q, E, R, L, H or K; (as) The amino acid residue at position 68 is K, R, E or N; (at) The amino acid residue at position 69 is Q or P; (au) The amino acid residue at position 79 is E or D; (av) The amino acid residue at position 80 is G or E; (aw) The amino acid residue at position 81 is Y, N or F; (ax) The amino acid residue at position 82 is R or H; (ay) The amino acid residue at position 83 is E, G or D; (az) The amino acid residue at position 84 is Q, R or L; (ba) Amino acid residue at position 86 is A or V; (bb) The amino acid residue at position 89 is T or S; (bc) The amino acid residue at position 90 is L or I; (bd) The amino acid residue at position 91 is I or V; (be) The amino acid residue at position 92 is R or K; (bf) The amino acid residue at position 93 is H, Y or Q; (bg) The amino acid residue at position 96 is E, A or Q; (bh) The amino acid residue at position 97 is L or I; (bi) The amino acid residue at position 100 is K, R, N or E; (bj) The amino acid residue at position 101 is K or R; (bk) The amino acid residue at position 103 is A or V; (bl) The amino acid residue at position 104 is D or N; The amino acid residue at position (bm) 105 is L or M; (bn) The amino acid residue at position 106 is L or I; (bo) The amino acid residue at position 112 is T or I; The amino acid residue at position (bp) 113 is S, T or F; (bq) The amino acid residue at position 114 is A or V; (br) The amino acid residue at position 115 is S, R or A; (bs) The amino acid residue at position 119 is K, E or R; (bt) The amino acid residue at position 120 is K or R; (bu) The amino acid residue at position 123 is F or L; (bv) The amino acid residue at position 124 is S or R; (bw) The amino acid residue at position 125 is E, K, G or D; The amino acid residue at position (bx) 126 is Q or H; The amino acid residue at position (by) 128 is E, G or K; The amino acid residue at position (bz) 129 is V, I or A; (ca) The amino acid residue at position 130 is Y, H, F or C; (cb) The amino acid residue at position 131 is D, G, N or E; The amino acid residue at position (cc) 132 is I, T, A, M, V or L; The amino acid residue at position (cd) 135 is V, T, A or I; (ce) The amino acid residue at position 138 is H or Y; (cf) The amino acid residue at position 139 is I or V; (cg) The amino acid residue at position 140 is L or S; The amino acid residue at position (ch) 142 is Y or H; (ci) The amino acid residue at position 143 is K, T or E; (cj) The amino acid residue at position 144 is K, E or R; (ck) The amino acid residue at position 145 is L or I; and (cl) The amino acid residue at position 146 is T or A,Polypeptide.(120) The isolated or recombinant polypeptide of item 42, wherein at least 80% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions: (a) Amino acid residues at positions 9, 76, 94 and 110 are A; (b) The amino acid residues at positions 29 and 108 are C; (c) The amino acid residue at position 34 is D; (d) The amino acid residue at position 95 is E; (e) The amino acid residue at position 56 is F; (f) The amino acid residues at positions 43, 44, 66, 74, 87, 102, 116, 122, 127 and 136 are G; (g) The amino acid residue at position 41 is H; (h) The amino acid residue at position 7 is I; (i) The amino acid residue at position 85 is K; (j) The amino acid residues at positions 20, 36, 42, 50, 72, 78, 98 and 121 are L; (k) The amino acid residues at positions 1, 75 and 141 are M; (l) The amino acid residues at positions 23, 64 and 109 are N; (m) The amino acid residues at positions 22, 25, 133, 134 and 137 are P; (n) The amino acid residue at position 71 is Q; (o) The amino acid residues at positions 16, 21, 73, 99 and 111 are R; (p) The amino acid residues at positions 55 and 88 are S; (q) The amino acid residue at position 77 is T; (r) The amino acid residue at position 107 is W; and (s) The amino acid residues at positions 13, 46, 70, 117 and 118 are Y,Polypeptide.(121) The isolated or recombinant polypeptide of item 119, wherein at least 90% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions: (a) 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121 and / Or the amino acid residue at position 141 is B1; (b) 16, 21, 22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108, 109 , 111, 116, 122, 127, 133, 134, 136 and / or the amino acid residue at position 137 is B2;Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y, and V; and B2 is R, N, D, C, Q, E, G, H, An amino acid selected from the group consisting of K, P, S and T,Polypeptide.(122) The isolated or recombinant polypeptide of item 120, wherein at least 90% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions: (a) Nos. 2, 4, 15, 19, 26, 28, 31, 45, 51, 54, 86, 90, 91, 97, 103, 105, 106, 114, 123, 129, 139 and / or 145. The amino acid residue is B1; (b) 3, 5, 8, 10, 11, 14, 17, 18, 24, 27, 32, 37, 38, 47, 48, 49, 52, 57, 58, 61, 62, 63, 68, 69 , 79, 80, 82, 83, 89, 92, 100, 101, 104, 119, 120, 124, 125, 126, 128, 131, 143 and / or the amino acid residue at position 144 is B2;Where B1 is an amino acid selected from the group consisting of A, I, L, M, F, W, Y, and V; and B2 is R, N, D, C, Q, E, G, H, An amino acid selected from the group consisting of K, P, S and T,Polypeptide.(123) At least 90% of the amino acid residues in the amino acid sequence corresponding to the following positions in the isolated or recombinant polypeptide of item 119 meet the following restrictions: (a) The amino acid residue at positions 1, 7, 9, 20, 36, 42, 50, 64, 72, 75, 76, 78, 94, 98, 110, 121 and / or 141 is Z1; (b) The amino acid residue at positions 13, 46, 56, 70, 107, 117 and / or 118 is Z2; (c) The amino acid residue at positions 23, 55, 71, 77, 88 and / or 109 is Z3; (d) The amino acid residue at positions 16, 21, 41, 73, 85, 99 and / or 111 is Z4; (e) The amino acid residue at position 34 and / or 95 is Z5; (f) The amino acid residue at positions 22, 25, 29, 43, 44, 66, 74, 87, 102, 108, 116, 122, 127, 133, 134, 136 and / or 137 is Z6;Where Z1 is an amino acid selected from the group consisting of A, I, L, M and V; Z2 is an amino acid selected from the group consisting of F, W and Y; Z3 is an amino acid selected from the group consisting of N, Q, S and Z4 is an amino acid selected from the group consisting of T; Z4 is an amino acid selected from the group consisting of R, H and K; Z5 is an amino acid selected from the group consisting of D and E; and Z6 is an amino acid selected from the group consisting of C. , An amino acid selected from the group consisting of G and P,Polypeptide.(124) The isolated or recombinant polypeptide of item 120, wherein at least 80% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions: (a) The amino acid residue at position 2, 4, 15, 19, 26, 28, 51, 54, 86, 90, 91, 97, 103, 105, 106, 114, 129, 139 and / or 145 is Z1. is there; (b) The amino acid residue at position 31 and / or 45 is Z2; (c) The amino acid residue at position 8 and / or 89 is Z3; (d) The amino acid residue at positions 82, 92, 101 and / or 120 is Z4; (e) The amino acid residue at positions 3, 11, 27 and / or 79 is Z5; (f) The amino acid residue at position 123 is Z1 or Z2; (g) The amino acid residues at positions 12, 33, 35, 39, 53, 59, 112, 132, 135, 140 and / or 146 are Z1 or Z3; (h) The amino acid residue at position 30 is Z1 or Z4; (i) The amino acid residue at position 6 is Z1 or Z6; (j) The amino acid residue at position 81 and / or 113 is Z2 or Z3; (k) The amino acid residue at position 138 and / or 142 is Z2 or Z4; (l) The amino acid residues at positions 5, 17, 24, 57, 61, 124 and / or 126 are Z3 or Z4; (m) The amino acid residue at position 104 is Z3 or Z5; (o) The amino acid residues at positions 38, 52, 62 and / or 69 are Z3 or Z6; (p) The amino acid residues at positions 14, 119 and / or 144 are Z4 or Z5; (q) The amino acid residue at position 18 is Z4 or Z6; (r) The amino acid residues at positions 10, 32, 48, 63, 80 and / or 83 are Z5 or Z6; (s) The amino acid residue at position 40 is Z1, Z2 or Z3; (t) The amino acid residues at positions 65 and / or 96 are Z1, Z3 or Z5; (u) The amino acid residue at position 84 and / or 115 is Z1, Z3 or Z4; (v) The amino acid residue at position 93 is Z2, Z3 or Z4; (w) The amino acid residue at position 130 is Z2, Z4 or Z6; (x) The amino acid residues at positions 47 and / or 58 are Z3, Z4 or Z6; (y) The amino acid residues at positions 49, 68, 100 and / or 143 are Z3, Z4 or Z5; (z) The amino acid residue at position 131 is Z3, Z5 or Z6; (aa) The amino acid residue at position 125 and / or 128 is Z4, Z5 or Z6; (ab) The amino acid residue at position 67 is Z1, Z3, Z4 or Z5; (ac) The amino acid residue at position 60 is Z1, Z4, Z5 or Z6; (ad) The amino acid residue at position 37 is Z3, Z4, Z5 or Z6;Where Z1 is an amino acid selected from the group consisting of A, I, L, M and V; Z2 is an amino acid selected from the group consisting of F, W and Y; Z3 is an amino acid selected from the group consisting of N, Q, S and Z4 is an amino acid selected from the group consisting of T; Z4 is an amino acid selected from the group consisting of R, H and K; Z5 is an amino acid selected from the group consisting of D and E; and Z6 is an amino acid selected from the group consisting of C. , An amino acid selected from the group consisting of G and P,Polypeptide.(125) The isolated or recombinant polypeptide of item 119, wherein at least 80% of the amino acid residues in the amino acid sequence corresponding to the following positions meet the following restrictions: (a) Amino acid residues at positions 9, 76, 94 and 110 are A; (b) The amino acid residues at positions 29 and 108 are C; (c) The amino acid residue at position 34 is D; (d) The amino acid residue at position 95 is E; (e) The amino acid residue at position 56 is F; (f) The amino acid residues at positions 43, 44, 66, 74, 87, 102, 116, 122, 127 and 136 are G; (g) The amino acid residue at position 41 is H; (h) The amino acid residue at position 7 is I; (i) The amino acid residue at position 85 is K; (j) The amino acid residues at positions 20, 36, 42, 50, 72, 78, 98 and 121 are L; (k) The amino acid residues at positions 1, 75 and 141 are M; (l) The amino acid residues at positions 23, 64 and 109 are N; (m) The amino acid residues at positions 22, 25, 133, 134 and 137 are P; (n) The amino acid residue at position 71 is Q; (o) The amino acid residues at positions 16, 21, 73, 99 and 111 are R; (p) The amino acid residues at positions 55 and 88 are S; (q) The amino acid residue at position 77 is T; (r) The amino acid residue at position 107 is W; and (s) The amino acid residues at positions 13, 46, 70, 117 and 118 are Y,Polypeptide.(126) The isolated or recombinant polypeptide according to item 24, wherein the amino acid residue in the amino acid sequence corresponding to position 28 here is V.(127) The isolated or recombinant polypeptide according to item 42, wherein the amino acid sequence is selected from the group consisting of SEQ ID NOs: 6-10 and 263-514.(128) A transgenic plant or transgenic plant explant having enhanced glyphosate resistance, wherein the plant or plant explant is glyphosate resistant by a polypeptide having glyphosate N-acetyltransferase activity and further mechanisms. A transgenic plant or transgenic plant explant that expresses at least one polypeptide that imparts.(129) The transgenic plant or transgenic plant explant according to item 128, wherein the polypeptide having glyphosate N-acetyltransferase activity is selected from the group consisting of SEQ ID NOs: 6-10 and 263-514. A transgenic plant or transgenic plant explant containing the amino acid sequence to be produced.(130) The transgenic plant or transgenic plant explant according to item 129, wherein at least one polypeptide conferring glyphosate resistance by a further mechanism is glyphosate resistant 5-enolpyrvir simimate-3. -Transgenic plants or transgenic plant explants selected from the group consisting of phosphate synthase and glyphosate-resistant glyphosate xidreductase.(131) The transgenic plant or transgenic plant explant according to item 130, wherein at least one polypeptide conferring glyphosate resistance by a further mechanism is glyphosate resistant 5-enolpyrvir simimate-3. -Transgenic plants or transgenic plant explants that are phosphate synthase.(132) The transgenic plant or transgenic plant explant according to item 130, wherein at least one polypeptide that imparts glyphosate resistance by a further mechanism here is glyphosate-resistant glyphosate oxidodreductase. Plant or transgenic plant explants.(133) A transgenic plant or transgenic plant explant, wherein the plant or plant explant confer resistance to a polypeptide having glyphosate N-acetyltransferase activity and additional herbicides. A transgenic plant or transgenic plant explant that expresses a peptide.(134) The transgenic plant or transgenic plant explant according to item 133, wherein the polypeptide having glyphosate N-acetyltransferase activity is selected from the group consisting of SEQ ID NOs: 6-10 and 263-514. A transgenic plant or transgenic plant explant containing the amino acid sequence to be produced.(135) The transgenic plant or transgenic plant explant according to item 134, wherein at least one polypeptide conferring resistance to additional herbicides is a mutated hydroxyphenylpyrvate dioxygenase, sulfonamide. Select from the group consisting of amide-resistant acetolacate synthase, sulfonamide-resistant acetohydroxyate synthase, imidazolinone-resistant acetolactic synthase, imidazolinone-resistant acetohydroxynate synthase, phosphinoslysin acetyltransferase and mutated protoporphyrinogen oxidase. Transgenic plants or explants of transgenic plants.(136) The transgenic plant or transgenic plant explant according to item 135, wherein at least one polypeptide conferring resistance to additional herbicides is the mutated hydroxyphenylpyrvate dioxygenase. , Transgenic plants or transgenic plant explants.(137) The transgenic plant or transgenic plant explant according to item 135, wherein at least one polypeptide conferring resistance to additional herbicides is a sulfonamide-resistant acetactate synthase, transgenic. Plant or transgenic plant explants.(138) The transgenic plant or transgenic plant explant according to item 135, wherein at least one polypeptide conferring resistance to additional herbicides is a sulfonamide-resistant acethydroxyate synthase, trans. Genetic or transgenic plant explants.(139) The transgenic plant or transgenic plant explant according to item 135, wherein at least one polypeptide conferring resistance to additional herbicides is transgenic, imidazolinone resistant acetactate synthase. Plant or transgenic plant explants.(140) The transgenic plant or transgenic plant explant according to item 135, wherein at least one polypeptide conferring resistance to the additional herbicide is an imidazolinone resistant acethydroxy acid synthase.(141) The transgenic plant or transgenic plant explant according to item 135, wherein the at least one polypeptide conferring resistance to the additional herbicide is a phosphinosricin acetyltransferase.(142) The transgenic plant or transgenic plant explant according to claim 135, wherein at least one polypeptide conferring resistance to the additional herbicide is a mutant protoporphyrinogen oxidase.(143) A transgenic plant or transgenic plant explant having enhanced resistance to glyphosate, wherein the plant or plant explant is a polypeptide having glyphosate N-acetyltransferase activity, by a further mechanism. A transgenic plant or transgenic plant explant that expresses at least one polypeptide that imparts resistance to glyphosate and at least one polypeptide that imparts resistance to additional herbicides.(144) The transgenic plant or transgenic plant according to item 143, wherein the polypeptide having glyphosate N-acetyltransferase activity comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 10 and 263 to 514. Plants.(145) The transgenic plant or transgenic plant explant according to item 144, wherein at least one polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant 5-enolpyrvir simimate-. At least one polypeptide selected from the group consisting of 3-phosphate synthase and glyphosate resistant glyphosate oxidoreductase and conferring resistance to said additional herbicides is a variant hydroxyphenylpyrvate dioxygenase, sulfonamide resistant acetolactate synthase, A transgenic plant selected from the group consisting of sulfonamide-resistant acetohydroxy acid synthase, imidazolinone-resistant acetolactate synthase, imidazolinone-resistant acetohydroxy acid synthase, phosphinosricin acetyltransferase, and mutant protoporphyrinogen oxidase. Transgenic plant explants.(146) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant 5-enolpyrvirsimimate-. A transgenic plant or transgenic plant explant, wherein the 3-phosphate synthase and at least one polypeptide conferring resistance to the additional herbicide is a variant hydroxyphenylpyrvate dioxygenase.(147) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant 5-enolpyrvirsimimate-. A transgenic plant or transgenic plant explant, wherein the 3-phosphate synthase and at least one polypeptide conferring resistance to the additional herbicide is a sulfonamide-resistant acetolactate synthase.(148) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant 5-enolpyrvirsimimate-. A transgenic plant or transgenic plant explant, wherein the 3-phosphate synthase and at least one polypeptide conferring resistance to the additional herbicide is a sulfonamide-resistant acethydroxyate synthase.(149) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant 5-enolpyrvir simimate-. A transgenic plant or transgenic plant explant, wherein the 3-phosphate synthase and at least one polypeptide conferring resistance to the additional herbicide is an imidazolinone-resistant acetactate synthase.(150) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant 5-enolpyrvir simimate-. A transgenic plant or transgenic plant explant, wherein the 3-phosphate synthase and at least one polypeptide conferring resistance to the additional herbicide is an imidazolinone-resistant acethydroxyate synthase.(151) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant 5-enolpyrvir simimate-. A transgenic plant or transgenic plant explant, wherein the 3-phosphate synthase and at least one polypeptide conferring resistance to the additional herbicide is phosphinosricin acetyltransferase.(152) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant 5-enolpyrvir simimate-. A transgenic plant or transgenic plant explant, wherein the 3-phosphate synthase and at least one polypeptide conferring resistance to the additional herbicide is a variant protoporphyrinogen oxidase.(153) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by said further mechanism is glyphosate resistant glyphosate oxidoreductase and. A transgenic plant or transgenic plant explant, wherein at least one polypeptide conferring resistance to the additional herbicide is a mutant hydroxyphenylpyrvate dioxygenase.(154) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by said further mechanism is glyphosate resistant glyphosate oxidoreductase and. A transgenic plant or transgenic plant explant, wherein at least one polypeptide conferring resistance to the additional herbicide is a sulfonamide-resistant acetolactate synthase.(155) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by said further mechanism is glyphosate resistant glyphosate oxidoreductase and. A transgenic plant or transgenic plant explant, wherein at least one polypeptide conferring resistance to the additional herbicide is a sulfonamide-resistant acethydroxyate synthase.(156) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by said further mechanism is glyphosate resistant glyphosate oxidoreductase and. A transgenic plant or transgenic plant explant, wherein at least one polypeptide conferring resistance to the additional herbicide is imidazolinone-resistant acetolactate synthase.(157) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by said further mechanism is glyphosate resistant glyphosate oxidoreductase and. A transgenic plant or a transgenic plant explant, wherein the at least one polypeptide conferring resistance to the additional herbicide is imidazolinone resistant acethydroxyate synthase.(158) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by said further mechanism is glyphosate resistant glyphosate oxidoreductase and. A transgenic plant or a transgenic plant explant, wherein at least one polypeptide conferring resistance to the additional herbicide is phosphinosricin acetyltransferase.(159) The transgenic plant or transgenic plant explant according to item 145, wherein at least one polypeptide conferring glyphosate resistance by said further mechanism is glyphosate resistant glyphosate oxidoreductase and. A transgenic plant or a transgenic plant explant, wherein the at least one polypeptide conferring resistance to the additional herbicide is a mutant protoporphyrinogen oxidase.(160) A transgenic plant or transgenic plant explant having enhanced resistance to glyphosate, wherein the plant or plant explant expresses a polypeptide having glyphosate N-acetyltransferase activity. , And at least one polypeptide selected from the group consisting of glyphosate-resistant 5-enolpyrvir simimate-3-phosphate synthase and glyphosate-resistant glyphosate oxidoreductase, transgenic plants or transgenic plant explants.(161) The transgenic plant or transgenic plant according to item 160, wherein the polypeptide having glyphosate N-acetyltransferase activity comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 10 and 263 to 514. Plants.(162) The transgenic plant or transgenic plant explant according to claim 161 wherein the at least one polypeptide is glyphosate-resistant 5-enolpyrvirsimimate-3-phosphate synthase.(163) The transgenic plant or transgenic plant explant according to item 161 wherein the at least one polypeptide is a glyphosate-resistant glyphosate oxidoreductase.(164) A transgenic plant or a transgenic plant explant, wherein the plant or plant explant expresses a polypeptide having glyphosate N-acetyltransferase activity, and at least one polypeptide is present. , Mutant hydroxyphenylpyrvate dioxygenase, sulfonamide-resistant acetolactic synthase, sulfonamide-resistant acetohydroxy acid synthase, imidazolinone-resistant acetolactic synthase, imidazolinone-resistant acetohydroxy acid synthase, phosphinosricin acetyltransferase, and variants A transgenic plant or a transgenic plant explant selected from the group consisting of protoporphyrinogen oxidase.(165) The transgenic plant or transgenic plant according to item 164, wherein the polypeptide having glyphosate N-acetyltransferase activity comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 10 and 263 to 514. Plants.(166) The transgenic plant or transgenic plant explant according to item 165, wherein the at least one polypeptide is a mutant hydroxyphenylpyrvate dioxygenase.(167) The transgenic plant or transgenic plant explant according to item 165, wherein the at least one polypeptide is a sulfonamide-resistant acetactate synthase.(168) The transgenic plant or transgenic plant explant according to item 165, wherein the at least one polypeptide is a sulfonamide-resistant acethydroxy acid synthase.(169) The transgenic plant or transgenic plant explant according to item 165, wherein the at least one polypeptide is an imidazolinone-resistant acetactate synthase.(170) The transgenic plant or transgenic plant explant according to item 165, wherein the at least one polypeptide is an imidazolinone-resistant acethydroxy acid synthase.(171) The transgenic plant or transgenic plant explant according to item 165, wherein the at least one polypeptide is a phosphinosricin acetyltransferase.(172) The transgenic plant or transgenic plant explant according to item 165, wherein the at least one polypeptide is a mutant protoporphyrinogen oxidase.(173) A transgenic plant or transgenic plant explant having enhanced resistance to glyphosate, wherein the plant or plant explant expresss a polypeptide having glyphosate N-acetyltransferase activity. , At least one first polypeptide is selected from the group consisting of glyphosate resistant 5-enolpyrvir simimate-3-phosphate synthase and glyphosate resistant glyphosate oxidoreductase, and at least one second polypeptide is mutated. Type hydroxyphenylpyrvate dioxygenase, sulfonamide-resistant acetolactic synthase, sulfonamide-resistant acetohydroxyate synthase, imidazolinone-resistant acetolactate synthase, imidazolinone-resistant acetohydroxynate synthase, phosphinosricin acetyltransferase and mutant protoporphylli A transgenic plant or a transgenic plant explant selected from the group consisting of nogen oxidase.(174) The transgenic plant or transgenic plant according to item 173, wherein the polypeptide having glyphosate N-acetyltransferase activity comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 10 and 263 to 514. Plants.(175) Item 174, wherein the first polypeptide is glyphosate-resistant 5-enolpyrvirsimimate-3-phosphate synthase, and the second polypeptide is a mutant hydroxyphenylpyrvate dioxygenase. Transgenic plants or transgenic plant explants.(176) The trans of item 174, wherein the first polypeptide is a glyphosate resistant 5-enolpyrvir simimate-3-phosphate synthase and the second polypeptide is a sulfonamide resistant acetractate synthase. Genetic or transgenic plant explants.(177) The first polypeptide is a glyphosate resistant 5-enolpyrvir silicate-3-phosphate synthase, and the second polypeptide is a sulfonamide resistant acethydroxy acid synthase, according to claim 174. Transgenic plants or transgenic plant explants.(178) The trans of item 174, wherein the first polypeptide is glyphosate resistant 5-enolpyrvir simimate-3-phosphate synthase, and the second polypeptide is imidazolinone resistant acetractate synthase. Genetic or transgenic plant explants.(179) The first polypeptide is glyphosate-resistant 5-enolpyrvirsimimate-3-phosphate synthase, and the second polypeptide is imidazolinone-resistant acetoxyacid synthase, claim 174. Transgenic plants or transgenic plant explants.(180) The first polypeptide is glyphosate-resistant 5-enolpyrvirsimimate-3-phosphate synthase, and the second polypeptide is phosphinosricin acetyltransferase, according to claim 174. Transgenic plants or explants of transgenic plants.(181) The first polypeptide is glyphosate-resistant 5-enolpyrvirsimimate-3-phosphate synthase, and the second polypeptide is a mutant protoporphyrinogen oxidase, item 174. Transgenic plant or transgenic plant explant.(182) The transgenic plant or transgenic plant according to item 174, wherein the first polypeptide is a glyphosate resistant glyphosate oxidoreductase and the second polypeptide is a mutant hydroxyphenylpyrvate dioxygenase. Explanted pieces.(183) The transgenic plant or transgenic plant explant according to item 174, wherein the first polypeptide is a glyphosate resistant glyphosate oxidoreductase and the second polypeptide is a sulfonamide resistant acetactate synthase. Piece.(184) The transgenic plant or transgenic plant explant according to item 174, wherein the first polypeptide is a glyphosate resistant glyphosate oxidoreductase and the second polypeptide is a sulfonamide resistant acethydroxy acid synthase. Piece.(185) The transgenic plant or transgenic plant explant according to item 174, wherein the first polypeptide is a glyphosate resistant glyphosate oxidoreductase and the second polypeptide is an imidazolinone resistant acetolactate synthase. ..(186) The transgenic plant or transgenic plant explant according to item 174, wherein the first polypeptide is a glyphosate resistant glyphosate oxidoreductase and the second polypeptide is an imidazolinone resistant acethydroxy acid synthase. Piece.(187) The transgenic plant or transgenic plant explant according to item 174, wherein the first polypeptide is a glyphosate resistant glyphosate oxidoreductase and the second polypeptide is a phosphinosricin acetyltransferase. Piece.(188) The transgenic plant or transgenic plant explant according to item 174, wherein the first polypeptide is a glyphosate resistant glyphosate oxidoreductase and the second polypeptide is a mutant protoporphyrinogen oxidase. Piece.(189) A transgenic plant or transgenic plant explant having enhanced resistance to glyphosate, wherein the plant or plant explant expresss a polypeptide having glyphosate N-acetyltransferase activity. , And at least one polypeptide is glyphosate-resistant 5-enolpyrvirsimimate-3-phosphate synthase, glyphosate-resistant glyphosate oxidoreductase, mutant hydroxyphenylpyruvate dioxygenase, sulfonamide-resistant acetactate synthase, sulfonamide-resistant. Transgenic or non-transgenic plants selected from the group consisting of acetohydroxyate synthase, imidazolinone-resistant acetactate synthase, imidazolinone-resistant acetohydroxyate synthase, phosphinosricin acetyltransferase and mutant protoporphyrinogen oxidase. Plants.(190) The transgenic plant or transgenic plant according to item 189, wherein the polypeptide having glyphosate N-acetyltransferase activity comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 10 and 263 to 514. Plants.(191) The transgenic plant or transgenic plant explant according to item 190, wherein the polypeptide is glyphosate resistant 5-enolpyrvir simimate-3-phosphate synthase.(192) The transgenic plant or transgenic plant explant according to item 190, wherein the polypeptide is a glyphosate-resistant glyphosate oxidoreductase.(193) The transgenic plant or transgenic plant explant according to item 190, wherein the polypeptide is a mutant hydroxyphenylpyrvate dioxygenase.(194) The transgenic plant or transgenic plant explant according to item 190, wherein the polypeptide is a sulfonamide-resistant acetactate synthase.(195) The transgenic plant or transgenic plant explant according to item 190, wherein the polypeptide is a sulfonamide-resistant acethydroxy acid synthase.(196) The transgenic plant or transgenic plant explant according to item 190, wherein the polypeptide is an imidazolinone-resistant acetactate synthase.(197), wherein the polypeptide is a imidazolinone-tolerant acetohydroxy acid synthase, bets of claim 190 transgenic plant or transgenic plant explant.(198) The transgenic plant or transgenic plant explant according to item 190, wherein the polypeptide is a phosphinosricin acetyltransferase.(199) The transgenic plant or transgenic plant explant according to item 190, wherein the polypeptide is a mutant protoporphyrinogen oxidase.(200) A method for controlling weeds in fields containing crops. (a) The step of planting a crop seed or plant in the field that has been transformed with a gene encoding glyphosate N-acetyltransferase and at least one gene encoding a polypeptide that imparts glyphosate resistance by a further mechanism. ;and, (b) The step of applying an effective application of glyphosate to crops and weeds in the field, which is sufficient to inhibit the growth of the weeds in the field without significant effects on the crop.Including, methods.(201) The method of item 200, wherein the gene encoding the glyphosate N-acetyltransferase comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 and 11-262.(202) The method according to item 201, wherein the polypeptide conferring glyphosate resistance by the further mechanism is from glyphosate-resistant 5-enolpyrvirsimimate-3-phosphate synthase and glyphosate-resistant glyphosate oxidoreductase. A method selected from the group of(203) The method of item 202, wherein the polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant 5-enolpyrvirsimimate-3-phosphate synthase.(204) The method of item 202, wherein the polypeptide that imparts glyphosate resistance by the additional mechanism is glyphosate-resistant glyphosate oxidoreductase.(205) A method for preventing the outbreak of glyphosate-resistant weeds in fields containing crops. (a) The step of planting a crop seed or plant in the field that has been transformed with a gene encoding glyphosate N-acetyltransferase and at least one gene encoding a polypeptide that imparts glyphosate resistance by a further mechanism. ;and, (b) The process of applying the effective application of glyphosate to crops and weeds in the field,Including, methods.(206) The method of item 205, wherein the gene encoding the glyphosate N-acetyltransferase comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 and 11-262.(207) The method of item 206, wherein the polypeptide conferring glyphosate resistance by the additional mechanism is from glyphosate-resistant 5-enolpyrvirsimimate-3-phosphate synthase and glyphosate-resistant glyphosate oxidoreductase. A method selected from the group of(208) The method of item 207, wherein the polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant 5-enolpyrvirsimimate-3-phosphate synthase.(209) The method of item 207, wherein the polypeptide that imparts glyphosate resistance by the additional mechanism is glyphosate-resistant glyphosate oxidoreductase.(210) A method for selectively controlling weeds in fields containing crops, such as: (a) The step of planting a crop seed or plant in the field that has been transformed with a gene encoding glyphosate N-acetyltransferase and at least one gene encoding a polypeptide that imparts resistance to additional herbicides. ,and; (b) Simultaneous or staggered application of glyphosate and additional herbicides in the field, sufficient to inhibit the growth of the weed in the field without significant impact on the crop. Process applied to,Including the process.(211) The method of item 210, wherein the gene encoding the glyphosate N-acetyltransferase comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 and 11-262.(212) The method of item 211, wherein at least one polypeptide conferring resistance to the additional herbicide is a variant hydroxyphenylpyrvate dioxygenase, sulfonamide resistant acetactate synthase, sulfonamide resistant. A method selected from the group consisting of acetohydroxy acid synthase, imidazolinone-resistant acetolactate synthase, imidazolinone-resistant acetohydroxy acid synthase, phosphinosricin acetyltransferase, and mutant protoporphyrinogen oxidase.(213) The method of item 211, wherein the additional herbicide is hydroxyphenylpyrvate dioxygenase inhibitor, sulfonamide, imidazolinone, bialaphos, phosphinosricin, azaphenidine, butafenacil, sulfosate, glufosinate. , And a method selected from the group consisting of protox inhibitors.(214) The method of item 212, wherein the polypeptide conferring resistance to the additional herbicide is a mutant hydroxyphenylpyrvate dioxygenase.(215) The method of item 212, wherein the polypeptide conferring resistance to the additional herbicide is a sulfonamide resistant acetactate synthase.(216) The method of item 212, wherein the polypeptide conferring resistance to the additional herbicide is a sulfonamide-resistant acethydroxy acid synthase.(217) The method of item 212, wherein the polypeptide conferring resistance to the additional herbicide is an imidazolinone-resistant acetactate synthase.(218) The method of item 212, wherein the polypeptide conferring resistance to the additional herbicide is an imidazolinone-resistant acethydroxy acid synthase.(219) The method of item 212, wherein the polypeptide conferring resistance to the additional herbicide is phosphinosricin acetyltransferase.(220) The method of item 212, wherein the polypeptide conferring resistance to the additional herbicide is a mutant protoporphyrinogen oxidase.(221) A method for preventing the outbreak of herbicide-tolerant weeds in fields containing crops, such as: (a) The step of planting a crop seed or plant in the field that has been transformed with a gene encoding glyphosate N-acetyltransferase and at least one gene encoding a polypeptide that imparts resistance to additional herbicides. ,and; (b) The step of applying simultaneous or staggered application of glyphosate and additional herbicides to crops and weeds in the field.Including, methods.(222) The method of item 221 wherein the gene encoding the glyphosate N-acetyltransferase comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 and 11-262.(223) The method of item 222, wherein at least one polypeptide conferring resistance to the additional herbicide is a variant hydroxyphenylpyrvate dioxygenase, sulfonamide resistant acetactate synthase, sulfonamide resistant. A method selected from the group consisting of acetohydroxy acid synthase, imidazolinone-resistant acetolactate synthase, imidazolinone-resistant acetohydroxy acid synthase, phosphinosricin acetyltransferase, and mutant protoporphyrinogen oxidase.(224) The method of item 221, wherein the additional herbicide is hydroxyphenylpyrvate dioxygenase inhibitor, sulfonamide, imidazolinone, bialaphos, phosphinosricin, azaphenidine, butafenacil, sulfosate, glufosinate. , And a method selected from the group consisting of protox inhibitors.(225) The method of item 223, wherein the polypeptide conferring resistance to the additional herbicide is a mutant hydroxyphenylpyrvate dioxygenase.(226) The method of item 223, wherein the polypeptide conferring resistance to the additional herbicide is a sulfonamide resistant acetactate synthase.(227) The method of item 223, wherein the polypeptide conferring resistance to the additional herbicide is a sulfonamide-resistant acethydroxy acid synthase.(228) The method of item 223, wherein the polypeptide conferring resistance to the additional herbicide is an imidazolinone-resistant acetactate synthase.(229) The method of item 223, wherein the polypeptide conferring resistance to the additional herbicide is an imidazolinone-resistant acethydroxy acid synthase.(230) The method of item 223, wherein the polypeptide conferring resistance to the additional herbicide is phosphinosricin acetyltransferase.(231) The method of item 223, wherein the polypeptide conferring resistance to the additional herbicide is a mutant protoporphyrinogen oxidase.(232) A method for selectively controlling weeds in fields containing crops, such as: (a) Using a gene encoding glyphosate N-acetyltransferase, at least one gene encoding a polypeptide that imparts resistance to glyphosate by a further mechanism, and at least one gene encoding a polypeptide that imparts resistance to additional herbicides. The process of planting a transformed crop seed or plant in the field, and; (b) Simultaneous or staggered application of glyphosate and additional herbicides in the field, sufficient to inhibit the growth of the weed in the field without significant impact on the crop. Process applied to,Including, methods.(233) The method of item 232, wherein the gene encoding the glyphosate N-acetyltransferase comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 and 11-262.(234) The transgenic plant or transgenic plant explant according to item 233, wherein at least one polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant 5-enolpyrvir simimate-. A transgenic plant or transgenic plant explant selected from the group consisting of 3-phosphate synthase and glyphosate-resistant glyphosate oxidoreductase.(235) The transgenic plant or transgenic plant explant according to item 234, wherein at least one polypeptide conferring glyphosate resistance by said further mechanism is glyphosate resistant 5-enolpyrvir simimate-3-phosphate synthase. ..(236) The transgenic plant or transgenic plant explant according to item 234, wherein at least one polypeptide conferring glyphosate resistance by the additional mechanism is glyphosate resistant glyphosate oxidoreductase.(237) The method of item 233, wherein at least one polypeptide conferring resistance to the additional herbicide is a variant hydroxyphenylpyrvate dioxygenase, sulfonamide resistant acetactate synthase, sulfonamide resistant. A method selected from the group consisting of acetohydroxy acid synthase, imidazolinone-resistant acetolactate synthase, imidazolinone-resistant acetohydroxy acid synthase, phosphinosricin acetyltransferase, and mutant protoporphyrinogen oxidase.(238) The method of item 233, wherein the additional herbicide is hydroxyphenylpyrvate dioxygenase inhibitor, sulfonamide, imidazolinone, bialaphos, phosphinosricin, azaphenidine, butafenacil, sulfosate, glufosinate. , And a method selected from the group consisting of protox inhibitors.(239) The method of item 237, wherein the polypeptide conferring resistance to the additional herbicide is a mutant hydroxyphenylpyrvate dioxygenase.(240) The method of item 237, wherein the polypeptide conferring resistance to the additional herbicide is a sulfonamide resistant acetactate synthase.(241) The method of item 237, wherein the polypeptide conferring resistance to the additional herbicide is a sulfonamide-resistant acethydroxy acid synthase.(242) The method of item 237, wherein the polypeptide conferring resistance to the additional herbicide is an imidazolinone-resistant acetactate synthase.(243) The method of item 237, wherein the polypeptide conferring resistance to the additional herbicide is an imidazolinone-resistant acethydroxy acid synthase.(244) The method of item 237, wherein the polypeptide conferring resistance to the additional herbicide is phosphinosricin acetyltransferase.(245) The method of item 237, wherein the polypeptide conferring resistance to the additional herbicide is a mutant protoporphyrinogen oxidase.(246) A method for controlling the outbreak of herbicide-tolerant weeds in fields containing crops, such as: (a) Using a gene encoding glyphosate N-acetyltransferase, at least one gene encoding a polypeptide that imparts resistance to glyphosate by a further mechanism, and at least one gene encoding a polypeptide that imparts resistance to additional herbicides. The process of planting a transformed crop seed or plant in the field, and; (b) The step of applying simultaneous or staggered application of glyphosate and additional herbicides to crops and weeds in the field.Including, methods.(247) The method of item 246, wherein the gene encoding the glyphosate N-acetyltransferase comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 and 11-262.(248) The method of item 247, wherein at least one polypeptide conferring resistance to the additional herbicide is a variant hydroxyphenylpyrvate dioxygenase, sulfonamide resistant acetactate synthase, sulfonamide resistant. A method selected from the group consisting of acetohydroxy acid synthase, imidazolinone-resistant acetolactate synthase, imidazolinone-resistant acetohydroxy acid synthase, phosphinosricin acetyltransferase, and mutant protoporphyrinogen oxidase.(249) The method of item 247, wherein the additional herbicide is hydroxyphenylpyrvate dioxygenase inhibitor, sulfonamide, imidazolinone, bialaphos, phosphinosricin, azaphenidine, butafenacil, sulfosate, glufosinate. , And a method selected from the group consisting of protox inhibitors.(250) The method of item 248, wherein the polypeptide conferring resistance to the additional herbicide is a mutant hydroxyphenylpyrvate dioxygenase.(251) The method of item 248, wherein the polypeptide conferring resistance to the additional herbicide is a sulfonamide resistant acetactate synthase.(252) The method of item 248, wherein the polypeptide conferring resistance to the additional herbicide is a sulfonamide-resistant acethydroxy acid synthase.(253) The method of item 248, wherein the polypeptide conferring resistance to the additional herbicide is an imidazolinone-resistant acetactate synthase.(254) The method of item 248, wherein the polypeptide conferring resistance to the additional herbicide is an imidazolinone-resistant acethydroxy acid synthase.(255) The method of item 248, wherein the polypeptide conferring resistance to the additional herbicide is phosphinosricin acetyltransferase.(256) The method of item 248, wherein the polypeptide conferring resistance to the additional herbicide is a mutant protoporphyrinogen oxidase.(Gist of the present invention) It is an object of the present invention to provide methods and reagents for making organisms (eg, plants) resistant to glyphosate. This object and other objects of the present invention are provided by one or more embodiments described below. One embodiment of the present invention provides a novel polypeptide referred to herein as a GAT polypeptide. GAT polypeptides are characterized by their structural similarity to each other (eg, with respect to sequence similarity when GAT polypeptides are aligned with each other). Some GAT polypeptides have the ability to catalyze glyphosate N-acetyltransferase activity, i.e., acetylation of glyphosate. Some GAT polypeptides can also catalyze the acetylation of glyphosate analogs and / or glyphosate metabolites (eg, aminomethylphosphonic acid). Novel polynucleotides, referred to herein as GAT polynucleotides, are also provided. GAT polynucleotides are characterized by their ability to encode GAT polypeptides. In some embodiments of the invention, GAT polynucleotides replace one or more parent codons with synonymous codons that are preferentially used in plants as compared to parent codons for better plant expression. It is operated by. In other embodiments, the GAT polynucleotide is modified by introducing a nucleotide sequence encoding an N-terminal chloroplast transport peptide. GAT polypeptides, GAT polynucleotides and glyphosate N-acetyltransferase activities are described in more detail below. The present invention further includes some fragments of GAT polypeptides and GAT polynucleotides described herein. The present invention includes non-native variants of the polypeptides and polynucleotides described herein, in which one or more amino acids of the polypeptide encoded herein are mutated. The present invention further provides nucleic acid constructs containing the polynucleotides of the invention. The construct can be a vector such as a plant transformation vector. In some aspects, the vectors belonging to the present invention contain T-DNA sequences. The construct may optionally contain regulatory sequences (eg, promoters) that are operably linked to the GAT polynucleotide, where the promoter is heterologous with respect to the polynucleotide and transformed by a nucleic acid construct. It is effective in causing sufficient expression of the polypeptide encoded to enhance the glyphosate resistance of the plant cells. In some aspects of the invention, the GAT polynucleotide functions as a selection marker (eg, in plants, bacteria, actinomycetes, yeasts, algae or other fungi). For example, organisms transformed with a vector containing a GAT polynucleotide selectable marker can be selected based on their ability to grow in the presence of glyphosate. The GAT marker gene can be used for selection or screening of transformed cells expressing this gene. The present invention further provides a vector overlaid with traits, i.e. a vector encoding GAT and also containing a second polynucleotide sequence. This second polynucleotide sequence encodes a second polypeptide sequence that imparts a detectable phenotypic trait in a cell or organism expressing a valid level of the second polypeptide. This detectable phenotypic trait can serve as a selectable marker (eg, by conferring herbicide resistance, conferring pest resistance, or providing some kind of visibility marker). In one embodiment, the invention provides a composition containing two or more polynucleotides of the invention. Compositions comprising two or more GAT polynucleotides or encoded polypeptides are characteristic of the present invention. In some cases, these compositions are, for example, a library of nucleic acids containing at least three or more of the above nucleic acids. Nucleic Acids of the Invention with Restricted Endonucleases, RNAse or DNAse, as well as compositions produced by incubation of the nucleic acids of the invention in the presence of deoxyribonucleotide triphosphates and nucleic acid polymerases (eg, thermostable nucleic acid polymerases). Compositions produced by digestion of the nucleic acid, or other fragmentation of its nucleic acid (eg, by mechanical shearing, chemical cleavage, etc.) are also features of the invention. Cells transduced with the vectors of the invention, or vectors incorporating the nucleic acids of the invention in other manners, are one aspect of the invention. In a preferred embodiment, the cells express the polypeptide encoded by the nucleic acid described above. In certain embodiments, the cell incorporating the nucleic acid of the invention is a plant cell. Transgenic plants, transgenic plant cells, and transgenic plant explants incorporating the nucleic acids of the invention are also features of the invention. In some embodiments, the transgenic plant, transgenic plant cell, or transgenic plant explant expresses an exogenous polypeptide having glyphosate N-acetyltransferase activity encoded by the nucleic acid of the invention. The present invention also provides transgenic seeds produced by the transgenic plants of the present invention. The present invention is further glyphosate resistant by a polypeptide having glyphosate N-acetyltransferase activity and another mechanism (eg, glyphosate resistant 5-enolpyrvir shikimate-3-phosphate synthase and / or glyphosate resistant glyphosate xidreductase). Provided is a transgenic plant or transgenic plant explants with enhanced resistance to glyphosate for the expression of the polypeptide that provides. In a further embodiment, the invention provides a transgenic plant or transgenic plant explants that have enhanced resistance to glyphosate as well as resistance to additional herbicides by expression of the polypeptide. In a further embodiment, the invention presents a polypeptide having enhanced resistance to glyphosate as well as glyphosate N-acetyltransferase activity, another mechanism (eg, glyphosate resistant 5-enolpyruvyl shikimate-3-phosphate synthase and /). Alternatively, a polypeptide that imparts glyphosate resistance by glyphosate-resistant glyphosate xidreductase, and a polypeptide that imparts resistance to additional herbicides (eg, mutated hydroxyphenylpyruvate dioxygenase, sulfonamide-resistant acetolactate synthase, sulfonamide resistance). Resistance to additional herbicides due to the expression of acetolactate synthase, imidazolinone-resistant acetolactate synthase, imidazolinone-resistant acetolactate synthase, phosphinosricin acetyltransferase and mutated protoporphyrinogen oxidase) Provided is a transgenic plant having or a transgenic plant explant. The present invention also presents enhanced resistance to glyphosate, as well as polypeptides having glyphosate N-acetyltransferase activity and polypeptides that confer resistance to additional herbicides (eg, mutated hydroxyphenylpyrupine dioxygenase, sulfonamides). Further due to the expression of resistant acetolactate synthase, sulfonamide resistant acetolactate synthase, imidazolinone resistant acetolactate synthase, imidazolinone resistant acetolactate synthase, phosphinosricin acetyltransferase and mutated protoporphyrinogen oxidase) Provided is a transgenic plant or transgenic plant explants that are resistant to herbicides. A method of producing a polypeptide of the invention by introducing a nucleic acid encoding the polypeptide of the invention into a cell, subsequently expressing it, and recovering them from the cell or medium is a feature of the invention. In a preferred embodiment, the cell expressing the polypeptide of the invention is a transgenic plant cell. Polyclonal anti-sera that react with antigens derived from SEQ ID NOs: 6-10 and 263-514 but do not react with naturally occurring related sequences (eg, peptides represented by the partial sequence of GenBank registration number CAA70664). Specific binding to the polypeptide produced by, and / or the antibody produced by administration of an antigen derived from any one or more of SEQ ID NOs: 6-10 and 263-514 and / or the aforementioned antibody. , And all antibodies that do not specifically bind to the naturally occurring polypeptide corresponding to GenBank Registration No. CAA70664 are characteristic of the present invention. Another aspect of the invention relates to a method of diversifying polynucleotides for producing novel GAT polynucleotides and GAT polypeptides by recombinant or mutating the nucleic acids of the invention in vitro or in vivo. In one embodiment, recombination produces a library of at least one recombinant GAT polynucleotide. This library generated as described above is an embodiment of the present invention as well as the cells constituting this library. Furthermore, a method of producing a modified GAT polynucleotide by mutation of the nucleic acid of the present invention is an embodiment of the present invention. Recombinant and mutated GAT polynucleotides and polypeptides produced by the methods of the invention are also embodiments of the invention. In some aspects of the invention, diversification is achieved using recombination, which can be achieved in vitro, in vivo, in silico, or a combination thereof. Some examples of diversification methods described in more detail below are the family shuffling method and the synthetic shuffling method. The present invention comprises transforming a plant or plant cell with a polynucleotide encoding glyphosate N-acetyltransferase and, if necessary, regenerating a transgenic plant from the transformed plant cell. Provided is a method for producing a transgenic plant or plant cell. In one aspect, the polynucleotide is a GAT polynucleotide, optionally from a bacterial source. In certain aspects of the invention, the method proliferates a transformed plant or plant cell with a concentration of glyphosate that inhibits the growth of a wild-type plant of the same species without inhibiting the growth of the transformed plant. Can include letting. The method produces transformed plants or plant cells or progeny of plants or plant cells in increasing concentrations of glyphosate and / or in glyphosate concentrations that are lethal to allogeneic wild plants or plant cells. May include growing. Glyphosate-resistant transgenic plants produced by this method can be propagated, for example, by mating the plant with a second plant, so that at least some of such mated offspring become glyphosate resistant. Shown. The present invention further aims to plant seeds or crops of glyphosate-tolerant crops on agricultural land as a result of transformation with a gene encoding glyphosate N-acetyltransferase, and to control weeds without significant effects on the crop. Provided are methods for selectively controlling weeds in crop-bearing farmlands, including the application of sufficient amounts of glyphosate to crops and weeds in crops. The present invention further develops glyphosate resistance by a gene encoding glyphosate N-acetyltransferase and another mechanism (eg, glyphosate-resistant 5-enolpyrvir simiate-3-phosphate synthase and / or glyphosate-resistant glyphosate xidreductase). As a result of transformation with the gene encoding the giving polypeptide, the seeds or crops of glyphosate-tolerant crops can be planted on the crop, and sufficient amount of glyphosate to control the weeds without significant impact on the crop. Provided are methods of controlling weeds in crops and preventing the emergence of glyphosate-resistant weeds in crops, including application to crops and weeds. In a further embodiment, the invention relates to a gene encoding glyphosate N-acetyltransferase, another mechanism (eg, glyphosate-resistant 5-enolpyrvir simiate-3-phosphate synthase and / or glyphosate-resistant glyphosate oxidodreductase). Genes encoding polypeptides that confer glyphosate resistance, and polypeptides that confer resistance to additional herbicides (eg, mutated hydroxyphenylpyruvate dioxygenase, sulfonamide-resistant acetolactate synthase, sulfonamide-resistant acetolactate synthase, Glyphosate-resistant crop seeds or crops as a result of transformation with genes encoding imidazolinone-resistant acetolactate synthase, imidazolinone-resistant acetoxylate synthase, phosphinosricin acetyltransferase and mutated protoporphyrinogen oxidase) Glyphosate and additional herbicides (eg, hydroxyphenylpyruvate dioxygenase inhibitor, sulfonamide, imidazolinone, biaraphos) to plant on farmland with Includes application of (bialaphos), phosphonosricin, azafenidin, butafenacil, sulfosate, glycosate, and protoporphyrinogen oxidase (protox) inhibitors) to crops and weeds on farmland. Provides a method of controlling weeds on farmland and preventing the emergence of glyphosate-resistant weeds on farmland containing crops. The present invention further relates to genes encoding glyphosate N-acetyltransferase and genes encoding polypeptides that confer resistance to additional herbicides (eg, mutated hydroxyphenylpyruvate dioxygenase, sulfonamide-resistant acetolactic synthase, sulfonamide). Glyphosate-resistant crop seeds as a result of transformation with resistant acetohydroxyate synthase, imidazolinone-resistant acetolactic synthase, imidazolinone-resistant acetohydroxyate synthase, phosphinosricin acetyltransferase and mutated protoporphyrinogen oxidase) Or planting plants on farmland, and sufficient amounts of glyphosate and additional herbicides (eg, hydroxyphenylpyruvate dioxygenase inhibitor, sulfonamide, imidazolinone, bialaphos) to control weeds without significant impact on crops. , Phosphonosricin, Azaphenidine, Butaphenacyl, Sulfoseto, Glyphosate, and Modified Protoporphyrinogen Oxidase Inhibitors) Methods and herbicide resistance for controlling weeds in crop-bearing farmlands. Provides a way to prevent the emergence of weeds. The invention further provides a method for the production of genetically transformed plants that are resistant to glyphosate, in which recombinant double-stranded DNA molecules are inserted into the plant cell genome. In this step, the recombinant double-stranded DNA molecule described above is (i) a promoter that functions to induce the production of RNA sequences in plant cells; (ii) a structural DNA that causes the generation of RNA sequences encoding GAT. Sequences; and (iii) contain 3'untranslated regions that function to add a stretch of polyadenyl nucleotides to the 3'end of the RNA sequence in plant cells; where the promoters are heterologous with respect to the structural DNA sequence and A step applied to induce sufficient expression of a polypeptide encoded to enhance glyphosate resistance of a plant cell transformed by a DNA molecule; a step of acquiring transformed plant cells; and against glyphosate. Includes the step of regenerating a genetically transformed plant with increased resistance from transformed plant cells. The present invention further presents a step of growing a glyphosate-tolerant crop as a result of transformation with a gene encoding glyphosate N-acetyltransferase under conditions such that the crop produces the crop; and harvesting the crop from the crop. Provided are methods for the production of crops, including the process. These methods often involve the application of concentrations of glyphosate that are effective in controlling weeds on crops. Exemplary crops include cotton, corn, and soybean. The present invention also provides a computer including a database consisting of sequence records having a string corresponding to SEQ ID NO: 1-514, a computer-readable medium, and a centralized system. The centralized system described above, along with each other and / or any additional nucleic acid or amino acid sequences, for selection, alignment, translation, reverse translation or browsing of any one or more strings corresponding to SEQ ID NOs: 1-514. Include one or more instruction sets as needed.

<figref num="1">Figure 1 depicts the N-acetylation of glyphosate catalyzed by glyphosate N-acetyltransferase (GAT).</figref><figref num="2">FIG. 2 illustrates the detection of N-acetylglyphosate produced by an exemplary Bacillus culture expressing native GAT activity by a mass spectrometer.</figref><figref num="3">Figure 3 tabulates the relative identities between GAT sequences isolated from different strains of bacteria and yit I from Bacillus subtilis.</figref><figref num="4">FIG. 4 is a map of the plasmid pMAXY2120 for expressing and purifying the GAT enzyme from E. coli cultures.</figref><figref num="5">FIG. 5 is the output of a mass spectrometer showing increased N-acetylglyphosate production over time in a typical GAT enzyme reaction mixture.</figref><figref num="6">Figure 6 shows the glyphosate KMIs a plot of kinetic data for the GAT enzyme, calculated as 2.9 mM.</figref><figref num="7">Figure 7 shows K for acetyl-CoA.MIs a plot of kinetic data derived from the data in Figure 6, calculated with 2 μM.</figref><figref num="8">FIG. 8 is a schematic diagram describing the degradation of glyphosate in soil via the AMPA pathway.</figref><figref num="9">FIG. 9 is a schematic diagram describing the sarcosine pathway of glyphosate degradation.</figref><figref num="10">Figure 10 shows the BLOSUM62 matrix.</figref><figref num="11">FIG. 11 is a map of plasmid pMAXY2190.</figref><figref num="12">FIG. 12 depicts a T-DNA construct with a gat selectable marker.</figref><figref num="13">FIG. 13 depicts a yeast expression vector with a gat selectable marker.</figref>

(Detailed discussion) The present invention relates to a novel class of enzymes exhibiting N-acetyltransferase activity. In one aspect, the invention relates to a novel class of enzymes capable of acetylating glyphosate and glyphosate analogs (eg, enzymes with glyphosate N-transferase (GAT) activity). The above enzymes are characterized by their ability to acetylate the secondary amines of the compound. In some aspects of the invention, the compound is a herbicide (eg, glyphosate) as outlined in FIG. The compound can also be a glyphosate analog or a metabolite by glyphosate degradation (eg, aminomethylphosphonic acid). Glyphosate acetylation is an important catalytic step in one metabolic pathway for glyphosate catabolism, but the enzymatic acetylation of glyphosate by naturally occurring enzymes, isolated enzymes, or recombinant enzymes. The conversion has not been described so far. Thus, the nucleic acids and polypeptides of the invention provide a new biochemical pathway for introducing herbicide resistance.

In one aspect, the invention provides a novel gene encoding a GAT polypeptide. Isolated GAT and recombinant GAT polynucleotides that correspond to naturally occurring polynucleotides, as well as recombinant and engineered (eg, diversified) GAT polypeptides, are characteristic of the invention. is there. GAT polynucleotides are exemplified by SEQ ID NOs: 1-5 and 11-262. Specific GAT polynucleotide and polypeptide sequences are provided as illustrations to aid in the description of the invention, and this is a range of GAT polynucleotide and polypeptide types described and / or claimed herein. Is not intended to be limited.

The present invention also presents new or improved properties described herein, such as improved and / or enhanced properties (eg, Km for altered glyphosate, increased rate of catalysis, increased stability, etc.) To generate and select a diversified library for producing additional GAT polynucleotides, including polynucleotides encoding GAT polypeptides having (based on the selection of polynucleotide components of the library for activity). Provide a method for. The above polynucleotides are particularly conveniently used in the production of glyphosate-resistant transgenic plants.

The GAT polypeptide of the present invention exhibits novel enzymatic activity. Specifically, enzymatic acetylation of the synthetic herbicide glyphosate has not been observed prior to the present invention. Thus, the polypeptides described herein (eg, exemplified by SEQ ID NOs: 6-10 and 263-514) are novel biochemicals for the detoxification of glyphosate that function in vivo (eg, in plants). Define chemical pathways.

Thus, the nucleic acids and polypeptides of the invention are of significance in the production of glyphosate-resistant plants by providing new nucleic acids, polypeptides, and biochemical pathways for the design of herbicide selectivity in transgenic plants. It has a deep importance.

(Definition) Before describing the invention in detail, it should be understood that the invention is not limited to specific compounds or biological systems, which can of course vary. It should also be understood that the terminology used herein is for the sole purpose of describing particular embodiments and is not intended to be limiting. As used herein and in the appended claims, the singular forms "a," "an," and "the" are used in multiple references unless their content expressly indicates otherwise. including. Thus, for example, the reference to "a device" includes a combination of two or more such devices, and the reference to "a gene fusion construct" refers to a mixture of constructs. Including, etc.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. All similar or equivalent methods and materials described herein may be used in the work for testing the present invention, and specific examples using suitable materials and methods are described herein. Described in the book.

In the description and claims of the present invention, the following terminology is used based on the definitions presented below.

For the purposes of the present invention, the term "glyphosate" results in the production of glyphosate anion in plants (including any salt thereof) and other forms of N-phosphonomethylglycine that are effective for any herbicide. Should be considered to include the form of. The term "glyphosate analog" refers to any structural analog of glyphosate that has the ability to inhibit EPSPS to a level where the glyphosate analog is effective for herbicide.

As used herein, the term "glyphosate N-acetyltransferase activity" or "GAT activity" refers to the ability to catalyze the acetylation of the secondary amine group of glyphosate, eg, as illustrated in FIG. To say. "Glyphosate N-acetyltransferase" or "GAT" is an enzyme that catalyzes the acetylation of the amine group of glyphosate, glyphosate analogs, and / or major metabolites of glyphosate (ie, AMPA or sarcosin). In some preferred embodiments of the invention, GAT can transfer an acetyl group from acetyl-CoA to a secondary amine of glyphosate and a primary amine of AMPA. The exemplary GATs described herein are active at pH 5-9 and have optimal activity within the pH 6.5-8.0 range. Activity is a variety of kinetic parameters well known in the art (eg, k).cat, KM, And kcat/ KM) Can be quantified. These kinetic parameters can be determined as described below in Example 7. A given polynucleotide or complementary polynucleotide can be determined from any particular nucleotide sequence.

The terms "polynucleotide", "nucleotide sequence" and "nucleic acid" depend on nucleotides (A, C, T, U, G, etc., or analogs of naturally occurring or artificial nucleotides) such as related contexts. Used to refer to polymers of DNA or RNA, or their representations (eg, strings).

Similarly, the "amino acid sequence" is a polymer of amino acids (protein, polypeptide, etc.) or a character string meaning an amino acid polymer, and is context-dependent. The terms "protein", "polypeptide" and "peptide" may be used interchangeably herein.

Polynucleotides, polypeptides or other components are usually "isolated" when they are partially or completely separated from their associated components (other proteins, nucleic acids, cells, synthetic reagents, etc.). A nucleic acid or polypeptide is "recombinant" if it is an artificial or engineered protein or nucleic acid, or if it is derived from an artificial or engineered protein or nucleic acid. For example, a polynucleotide that is inserted into a vector or any other heterologous position (eg, in the genome of a living modified organism) and, as a result, is naturally found and is not associated with a nucleotide sequence normally adjacent to that polynucleotide. , Recombinant polynucleotide. Proteins expressed from recombinant polynucleotides in vitro or in vivo are examples of recombinant polypeptides. Similarly, non-naturally occurring polynucleotide sequences (eg, variants of naturally occurring genes) are recombinant.

The terms "glyphosate N-acetyltransferase polypeptide" and "GAT polypeptide" are used interchangeably to refer to any of the novel family of polypeptides provided herein.

The terms "glyphosate N-acetyltransferase polynucleotide" and "GAT polynucleotide" are used interchangeably to refer to a polynucleotide encoding a GAT polypeptide.

A "subsequence" or "fragment" is any part of the entire sequence.

Amino acid polymers or nucleotide polymers are numbered when the position of a given monomeric component of the polymer (amino acid residue, incorporated nucleotide, etc.) corresponds to the same residue position in the selected reference polypeptide or polynucleotide. Corresponds to the numbering of selected amino acid polymers or nucleic acids.

The vector is a composition for promoting cell transduction by the selected nucleic acid or expression of the nucleic acid in the cell. Examples of the vector include plasmids, cosmids, viruses, YACs, bacteria, polylysines, chromosomal integration vectors, episomal vectors and the like.

"Substantial overall length of a polynucleotide or amino acid sequence" means at least about 70% of the sequence, generally at least about 80% of the sequence, or typically about 90% or more of the sequence.

As used herein, "antibody" refers to a protein that comprises one or more polypeptides that are substantially or partially encoded by an immunoglobulin gene or an immunoglobulin gene fragment. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes, as well as innumerable immunoglobulin variable region genes. Light chains are classified as either κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin class, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kD) and one "heavy chain" (about 50 kD-70 kD). .. The N-terminus of each strand defines a variable region of about 100 to 110 or more amino acids that are primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light chains and heavy chains, respectively. Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin is an antibody under a disulfide bond in the hinge region to produce a dimer (F (ab) '2) of Fab, which is a light chain that itself binds to VH-CH1 by a disulfide bond. Digest. F (ab) '2 can reduce and break disulfide bonds in the hinge region under mild conditions, thereby converting the (Fab') 2 dimer to a Fab'monomer. Fab'monomers are effectively Fabs that have part of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, 4th Edition, WE Paul (eds.), Raven Press, NY. (1998)). Various antibody fragments are defined by digestion of intact antibodies, but those skilled in the art will appreciate that such Fab'fragments can be newly synthesized either chemically or by using recombinant DNA methodologies. To understand the. Therefore, the term antibody, as used herein, is also an antibody fragment that is either produced by modification of all antibodies or newly synthesized using recombinant DNA methodology. Include. Antibodies include single chain antibodies (including single chain Fv (sFv) antibodies in which variable heavy and variable light chains are linked together to form a contiguous polypeptide (either directly or through a peptide linker)). Can be mentioned.

A "chloroplast transit peptide" is an amino acid sequence that binds to and translates into a protein, directing the protein to the chloroplast or other pigment form present in the cell from which the protein is produced. "Chloroplast translocation sequence" refers to a nucleotide sequence encoding a chloroplast translocation peptide.

A "signal peptide" is an amino acid sequence that is translated by binding to a protein and directs the protein to the secretory system (Chrispeels, JJ, (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21. -53). If the protein is directed to the vacuole, a vacuolar targeting signal (above) can be added, or if directed to the endoplasmic reticulum, an endoplasmic reticulum retention signal (above) can be added. When a protein is directed to the nucleus, any signal peptide present should be removed and replaced with a nuclear localization signal (Raikhel, N. (1992) Plant Phys. 100: 1627-1632).

The terms "diversification" and "diversity" as applied to a polynucleotide refer to the generation of multiple variants of a parent polynucleotide, or the generation of multiple variants of a plurality of parent polynucleotides. When a polynucleotide encodes a polynucleotide, the diversity in the nucleotide sequence of the polynucleotide is that the corresponding polynucleotide being encoded has diversity (eg, a diverse pool of polynucleotides encoding multiple polypeptide variants). Can be produced. In some embodiments of the invention, this sequence diversity is live for diversified polynucleotides (eg, polynucleotides encoding GAT polypeptides with enhanced functional properties) for variants with the desired functional attributes. Obtained by screening / selecting a rally.

The term "encoding" refers to the ability of a nucleotide sequence to encode one or more amino acids. The term does not require a start codon or a stop codon. The amino acid sequence can be encoded in any one of the six different reading frames provided by the polynucleotide sequence and its complementary strands.

As used herein, the term "artificial variant" is a modified GAT polynucleotide (eg, SEQ ID NOs: 1-5 and 11-262, or naturally occurring isolated from an organism. A polypeptide having GAT activity, encoded by a modified form of any one of the GAT polynucleotides. The modified polynucleotide (which, when expressed in a suitable host, an artificial variant is produced from it) is obtained through human intervention by modifying the GAT polynucleotide.

The term "nucleic acid construct" or "polynucleotide construct" means either a single-stranded or double-stranded nucleic acid molecule, which is isolated from a naturally occurring gene or otherwise naturally occurring. It has been modified to contain a segment of nucleic acid in a manner that does not. The term nucleic acid construct is synonymous with the term "expression cassette" if the nucleic acid construct contains a regulatory sequence required for expression of the coding sequences of the invention.

The term "control sequence" is defined herein to include all components necessary or advantageous for the expression of the polypeptides of the invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. The control sequence includes, at a minimum, the promoter as well as the transcription termination signal and the translation termination signal. The control sequence may comprise a linker for introducing a specific restriction site that facilitates ligation of the control sequence with the coding region of the nucleotide sequence encoding the polypeptide.

The term "operably linked" is used herein as a configuration in which a control sequence is appropriately located at a position corresponding to the coding sequence of a DNA sequence so that the control sequence induces expression of a polypeptide. Defined as an array.

As used herein, the term "coding sequence" is intended to cover a nucleotide sequence that directly identifies the amino acid sequence of the protein product. The boundaries of the coding sequence are generally determined by the open reading frame, which usually begins at the ATG start codon. The coding sequence typically encloses DNA, cDNA, and / or recombinant nucleotide sequences.

In this context, the term "expression" includes, but is limited to, any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Not done.

In this context, the term "expression vector" covers a linear or cyclic DNA molecule that comprises a segment encoding a polypeptide of the invention, which is operably linked to a further segment that provides its transcription. To do.

As used herein, the term "host cell" includes any cell type that is susceptible to transformation with nucleic acid constructs.

The term "plant" refers to whole plants, seedling-like vegetative organs / structures (eg, leaves, stems, and stalks), roots, flowers and flower organs / structures (eg, ovules, pistils, petals, ovules, carpels, etc. Potatoes and ovules), seeds (including embryos, endomilk, and seed coat) and fruits (mature ovary), plant tissues (eg, vascular tissue, basal tissue, etc.) and cells (eg, guard cells, egg cells, etc.) (Protrusion-like structures, etc.), as well as their descendants. The classes of plants that can be used in the methods of the invention are generally susceptible to transformation techniques including anthropogenic plants (monocotyledonous and diploid plants), nude plants, ferns, and polyploid algae. The higher and lower plant classes are broader and include various polyploid levels of plants, including haploids, polyploids, diploids, haploids and semijunctions.

As used herein, the term "heterogeneous" describes a relationship between two or more elements that indicate that one element is in close proximity to the other and is not normally found in nature. Thus, for example, if the polynucleotide sequence is derived from an alien species, or if the polynucleotide sequence from the same species has been altered from its original form, then the polynucleoti sequence becomes an organism or a second polynucleotide sequence. On the other hand, it is "heterogeneous". For example, a promoter operably linked to a heterologous coding sequence is a coding sequence derived from a species different from the species from which the promoter is derived, or if it is a coding sequence derived from the same species, it is naturally present in the promoter. Refers to a coding sequence that is not related to (eg, a genetically engineered coding sequence or an allelic gene from a different ecosystem or variant). Examples of heterologous polypeptides include polypeptides expressed from recombinant polynucleotides in transgenic organisms. Heterologous polynucleotides and heterologous polypeptides take the form of recombinant molecules.

Various additional terms are defined herein or otherwise characterized. (Glyphosate N-acetyltransferase) In one aspect, the invention presents a novel family of isolated enzymes or recombinant enzymes (referred to herein as "glyphosate N-acetyltransferases", "GATs", or "GAT enzymes"). provide. GAT is an enzyme having GAT activity, preferably an enzyme having sufficient activity to impart some degree of glyphosate resistance to transgenic plants engineered to express GAT. Some examples of GAT include GAT polypeptides described in more detail below.

Naturally, GAT-mediated glyphosate resistance is a complex function of GAT activity, GAT expression levels in transgenic plants, specific plants, the nature and timing of herbicide application. One of ordinary skill in the art can determine the level of GAT activity required to result in glyphosate resistance in a particular situation without undue experimentation.

GAT activity is a customary dynamic parameter, kcat, KM, And kcat/ KMCan be characterized using. kcatIs considered as a measure of the rate of acetylation, especially at high substrate concentrations, KMIs a measure of the affinity of GAT for its substrates (eg, acetyl-CoA and glyphosate), and kcat/ KMIs a measure of catalytic effect that takes into account both substrate affinity and catalytic rate (this parameter is especially important in situations where the substrate concentration is at least partly rate-determining step). Generally higher kcatOr kcat/ KMGAT with lower kcatOr kcat/ KMIt is a more efficient catalyst than another GAT with. Lower KMGAT with higher KMIt is a more efficient catalyst than another GAT with. Therefore, one of ordinary skill in the art can compare the dynamic parameters of the two enzymes to determine if one GAT is more efficient than the other. kcat, Kcat/ KMAnd KMThe relative importance of is the situation in which GAT is expected to work (eg, K for glyphosate).MIt changes depending on the expected effective concentration of glyphosate). GAT activity can also be characterized by any of a number of functional properties, such as stability, susceptibility to inhibition, or activation by other molecules.

(Glyphosate N-acetyltransferase polypeptide) In one aspect, the invention presents a novel family of isolated or recombinant polypeptides (referred to herein as "glyphosate N-acetyltransferase polypeptides" or "GAT polypeptides"). provide. GAT polypeptides are characterized by their structural similarity to a novel family of GATs. Many, but not all, GAT polypeptides are GATs. Its properties are that GAT polypeptides are defined by structure, whereas GAT is defined by function. Some GAT polypeptides preferably comprise a GAT polypeptide having a level of GAT activity that functions to confer glyphosate resistance to transgenic plants expressing this protein at effective levels. Some preferred GAT polypeptides for use in conferring glyphosate resistance are at least 1 min.-1Kcat, Or more preferably at least 10 min-1, 100min-1, Or 1000min-1KcatHave. Another preferred GAT polypeptide used to confer glyphosate resistance is a K of around 100 mM.M, Or more preferably 10 mM, 1 mM or 0.1 mM or less KMHave. Yet another preferred GAT polypeptide for use in conferring glyphosate resistance is at least 1 mM.-1min-1Above kcat/ KM, Or more preferably at least 10 mM-1min-1, 100mM-1min-1, 1000mM-1min-1, Or 10,000 mM-1min-1Kcat/ KMHave.

Representative GAT polypeptides have been isolated and characterized from various bacterial strains. One example of an isolated and characterized monomeric GAT polypeptide has a molecular radius of approximately 17 kD. A representative GAT enzyme (SEQ ID NO: 7) isolated from B. licheniformis has a K of approximately 2.9 mM for glyphosate.mShows about 2 μM K for acetyl-CoAmIndicates (6 min-1KcatHas).

The term "GAT polypeptide" refers to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10 and 263-514, using the BLOSUM62 matrix, 11 gap presence penalties, and one gap extension penalty, and at least 430. Any polypeptide that contains an amino acid sequence that can be optimally aligned to produce a similarity score. Several aspects of the invention use the BLOSUM62 matrix, 11 gap presence penalties, one gap extension penalty, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10 and 263-514, and at least 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, To a GAT polypeptide containing an amino acid sequence that can be optimally aligned to produce a similarity score of 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, or 760. Related.

One aspect of the invention comprises an amino acid sequence that can be optimally aligned with SEQ ID NO: 457 to produce a similarity score of at least 430 using the BLOSUM62 matrix, 11 gap presence penalties, and 1 gap extension penalty. , Related to GAT polypeptides. Several aspects of the invention use the BLOSUM62 matrix, 11 gap presence penalties, and one gap extension penalty with SEQ ID NO: 457 and at least 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, It is associated with a GAT polypeptide that contains an amino acid sequence that can be optimally aligned to produce a similarity score of 730, 735, 740, 745, 750, 755, or 760.

One aspect of the invention comprises an amino acid sequence that can be optimally aligned with SEQ ID NO: 445 to produce a similarity score of at least 430 using the BLOSUM62 matrix, 11 gap presence penalties, and 1 gap extension penalty. , Related to GAT polypeptides. Several aspects of the invention use the BLOSUM62 matrix, 11 gap presence penalties, and one gap extension penalty with SEQ ID NO: 445 and at least 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, It is associated with a GAT polypeptide that contains an amino acid sequence that can be optimally aligned to produce a similarity score of 730, 735, 740, 745, 750, 755, or 760.

One aspect of the invention comprises an amino acid sequence that can be optimally aligned with SEQ ID NO: 300 to produce a similarity score of at least 430 using the BLOSUM62 matrix, 11 gap presence penalties, and 1 gap extension penalty. , Related to GAT polypeptides. Several aspects of the invention use the BLOSUM62 matrix, 11 gap presence penalties, and one gap extension penalty with SEQ ID NO: 300 and at least 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, It is associated with a GAT polypeptide that contains an amino acid sequence that can be optimally aligned to produce a similarity score of 730, 735, 740, 745, 750, 755, or 760.

The two sequences use a defined amino acid substitution matrix (eg, BLOSUM62), a gap presence penalty, and a gap extension penalty and they are used for similarity scoring to reach the highest possible score for that pair of sequences. When aligning, it is "optimally aligned". Amino acid substitution matrices and their use in quantifying similarities between two sequences are well known in the art, for example, "Atlas of Protein sequence and Structure" Vol. 5, Addendum 3 (MODayhoff), p. 345. -Page 352, Natl.Biomed.Res.Found, Dayhoff et al. (1978) "A model of evolutionary change in proteins." In Washington, DC, and Henikoff et al. (1992) Proc.Natl.Acad.Sci.USA 89:10 915 -10919. BLOSUM62 matrix (Figure 10) is often Gapped BLAST Used as a default scoring substitution matrix in sequence alignment protocols such as 2.0. The gap presence penalty imposes the introduction of one amino acid gap in one of the aligned sequences, and the gap extension penalty imposes each additional empty amino acid position inserted into the already opened gap. Alignment is defined by the amino acid position of each sequence from the beginning to the end of the alignment, and if necessary, one or more gaps in one or both sequences to reach the highest possible score. Specified by insertion. While optimal alignment and scoring can be achieved manually, this process is described in computer-executed alignment algorithms (eg, Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, National Center for Biotechnology Information Website ( Publicly available gapped BLAST at http://www.ncbi.nlm.nih.gov) Promoted by the use of 2.0). Optimal alignments, including multiple alignments, are available, for example, through http://www.ncbi.nlm.nih.gov and are described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. -Can be trimmed using BLAST.

For an amino acid sequence that is optimally aligned with the reference sequence, the amino acid residue "corresponds" to the position of the reference sequence in which the residue is paired in the alignment. The "position" is indicated by a number that continuously identifies each amino acid in the reference sequence based on its position relative to the N-terminus. For example, in SEQ ID NO: 300, position 1 is M, position 2 is I, position 3 is E, and so on. If a test sequence is optimally aligned with SEQ ID NO: 300, the residue of the test sequence that aligns with E at position 3 is said to "correspond to position 3" of SEQ ID NO: 300. For deletions, insertions, shortenings, fusions, etc. that should be taken into account when determining the optimal alignment, the amino acid residue number in the test sequence, which is generally determined simply by counting from the N-terminus, is the reference sequence. It does not have to be the same as the corresponding position number in. For example, if there is a deletion in the aligned test sequence, there is no amino acid corresponding to the position in the reference sequence at the deletion site. If there is an insertion in the aligned reference sequence, the insertion does not correspond to any amino acid position in the reference sequence. In the case of shortening or fusion, there may be a stretch of amino acids in either the reference sequence or the aligned sequence that does not correspond to any amino acid in the corresponding sequence.

The term "GAT polypeptide" further relates to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with respect to the amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10 and 263-514. Some aspects of the invention are at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97 with respect to amino acids selected from the group consisting of SEQ ID NOs: 6-10 and 263-514. It is related to a GAT polypeptide containing an amino acid sequence having a%, 98%, or 99% sequence identity.

One aspect of the invention relates to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with respect to SEQ ID NO: 457. Some aspects of the invention have at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 457. It is related to a GAT polypeptide that contains an amino acid sequence.

One aspect of the invention relates to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with respect to SEQ ID NO: 445. Some aspects of the invention have at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 445. It is related to a GAT polypeptide that contains an amino acid sequence.

One aspect of the invention relates to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with respect to SEQ ID NO: 300. Some aspects of the invention have at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 300. It is related to a GAT polypeptide that contains an amino acid sequence.

The term "GAT polypeptide" further refers to any poly comprising an amino acid sequence having at least 40% sequence identity for 1-96 residues of an amino acid selected from the group consisting of SEQ ID NOs: 6-10 and 263-514. Refers to peptides. Some aspects of the invention are at least 60%, 70%, 80%, 90%, 92%, 95 with respect to 1-96 residues of amino acids selected from the group consisting of SEQ ID NOs: 6-10 and 263-514. It is related to a GAT polypeptide containing an amino acid sequence having a%, 96%, 97%, 98%, or 99% sequence identity.

One aspect of the invention relates to a polypeptide comprising an amino acid sequence having at least 40% sequence identity with respect to residues 1-96 of SEQ ID NO: 457. Some aspects of the invention are at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% for residues 1-96 of SEQ ID NO: 457. It is related to a GAT polypeptide containing an amino acid sequence having the sequence identity of.

One aspect of the invention relates to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with respect to residues 1-96 of SEQ ID NO: 445. Some aspects of the invention are at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% for residues 1-96 of SEQ ID NO: 445. It is related to a GAT polypeptide containing an amino acid sequence having the sequence identity of.

One aspect of the invention relates to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with respect to residues 1-96 of SEQ ID NO: 300. Some aspects of the invention are at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% for residues 1-96 of SEQ ID NO: 300. It is related to a GAT polypeptide containing an amino acid sequence having the sequence identity of.

The term "GAT polypeptide" further refers to any poly comprising an amino acid sequence having at least 40% sequence identity for 51-146 residues of an amino acid selected from the group consisting of SEQ ID NOs: 6-10 and 263-514. Refers to peptides. Some aspects of the invention are at least 60%, 70%, 80%, 90%, 92%, 95 with respect to 51-146 residues of amino acids selected from the group consisting of SEQ ID NOs: 6-10 and 263-514. It is related to a polypeptide containing an amino acid sequence having a%, 96%, 97%, 98%, or 99% sequence identity.

One aspect of the invention relates to a polypeptide comprising an amino acid sequence having at least 40% sequence identity with respect to residues 51-146 of SEQ ID NO: 457. Some aspects of the invention are at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% for residues 51-146 of SEQ ID NO: 457. It is related to a GAT polypeptide containing an amino acid sequence having the sequence identity of.

One aspect of the invention relates to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with respect to residues 51-146 of SEQ ID NO: 445. Some aspects of the invention are at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% for residues 51-146 of SEQ ID NO: 445. It is related to a GAT polypeptide containing an amino acid sequence having the sequence identity of.

One aspect of the invention relates to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with respect to residues 51-146 of SEQ ID NO: 300. Some aspects of the invention are at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% for residues 51-146 of SEQ ID NO: 300. It is related to a GAT polypeptide containing an amino acid sequence having the sequence identity of.

As used herein, the term "identity" or "percentage of identity" is a Clustal W analysis available from the European Bioinformatics Institute, Cambridge, UK, when used with respect to a particular pair of aligned amino acid sequences. (Version W1.8) (counting the number of identical fits in the alignment, such a number of identical fits by the maximum (i) aligned sequence length, and the maximum (ii) 96 length. The following default ClustalW parameters to divide and achieve slow / accurate pairwise alignment: Gap open penalty: 10; Gap extension penalty: 10.10; Protein weight matrix: Gonnet series; DNA weight matrix: IUB; Toggle Slow / Fast pairwise Alignment = Amino acid sequence identity percentage obtained by (using SLOW or FULL Alignment).

In another aspect, the invention simply comprises at least 20, or 50, 75, 100, 125 or 140 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10 and 263-514. The separated polypeptide or recombinant polypeptide is provided.

In another aspect, the invention provides an isolated or recombinant polypeptide comprising at least 20, or 50, 100, or 140 contiguous amino acids of SEQ ID NO: 457.

In another aspect, the invention provides an isolated or recombinant polypeptide comprising at least 20, or 50, 100, or 140 contiguous amino acids of SEQ ID NO: 445.

In another aspect, the invention provides an isolated or recombinant polypeptide comprising at least 20, or 50, 100, or 140 contiguous amino acids of SEQ ID NO: 300.

In another aspect, the invention provides a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514.

Some preferred GAT polypeptides of the invention are characterized as follows. If desired, at least 90% of the amino acid residues corresponding to the following positions in the polypeptide when aligned with a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514. Is subject to the following constraints: (a) 2nd, 4th, 15th, 19th, 26th, 28th, 31st, 45th, 51st, 54th, 86th, 90th, 91st, 97th At positions 103, 105, 106, 114, 123, 129, 139, and / or 145, the amino acid residue is B1; and (b) positions 3, 5, and 8. , 10th, 11th, 14th, 17th, 18th, 24th, 27th, 32nd, 37th, 38th, 47th, 48th, 49th, 52nd, 57th, 58th, 61st 6th, 62nd, 63rd, 68th, 69th, 79th, 80th, 82nd, 83rd, 89th, 92nd, 100th, 101st, 104th, 119th, 120th, 124th, At positions 125, 126, 128, 131, 143, and / or 144, the amino acid residue is B2; where B1 is A, I, L, M, F, W, Y, and. It is an amino acid selected from the group consisting of V; B2 is an amino acid selected from the group consisting of R, N, D, C, Q, E, G, H, K, P, S, and T. When used to identify an amino acid or amino acid residue, the one-letter notation A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y have standard meanings as used in the art and as provided in Table 2 herein.

Some preferred GAT polypeptides of the invention are characterized as follows. If desired, at least 80% of the amino acid residues corresponding to the following positions in the polypeptide are aligned with the reference amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514. , (A) 2nd, 4th, 15th, 19th, 26th, 28th, 51st, 54th, 86th, 90th, 91st, 97th, 103rd, 105th , 106, 114, 129, 139, and / or 145, the amino acid residue is Z1; (b) at 31 and / or 45, the amino acid residue is Z2; (c) ) At positions 8 and / or 89, the amino acid residue is Z3; (d) at positions 82, 92, 101, and / or 120, the amino acid residue is Z4; (e) at position 3 , 11th, 27th, and / or 79th, the amino acid residue is Z5; (f) 123rd, the amino acid residue is Z1 or Z2; (g) 12th, 33rd, 35th. , 39, 53, 59, 112, 132, 135, 140, and / or 146, where the amino acid residue is Z1 or Z3; (h) at position 30, the amino acid residue is Z1 or Z4; (i) at position 6 and the amino acid residue is Z1 or Z6; (j) at position 81 and / or 113 and the amino acid residue is at position Z2 or Z3; (k) at position 138. And / or at position 142, the amino acid residues are Z2 or Z4; (l) at positions 5, 17, 24, 57, 61, 124, and / or 126, the amino acid residues are Z3 or Z4. At (m) 104, the amino acid residue is Z3 or Z5; (o) at 38, 52, 62, and / or 69, the amino acid residue is Z3 or Z6; ( p) At positions 14, 119 and / or 144, the amino acid residue is Z4 or Z5; (q) At position 18, the amino acid residue is Z4 or Z6;

Some preferred GAT polypeptides of the invention are characterized as follows. If desired, at least 90% of the amino acid residues corresponding to the following positions in the polypeptide are aligned with the reference amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514. , (A) 1st, 7th, 9th, 20th, 36th, 42nd, 50th, 64th, 72nd, 75th, 76th, 78th, 94th, 98th , 110, 121, and / or 141, the amino acid residue is Z1; (b) at 13, 46, 56, 70, 107, 117, and / or 118, The amino acid residue is Z2; (c) at positions 23, 55, 71, 77, 88, and / or 109, and the amino acid residue is Z3; (d) at positions 16, 21, At positions 41, 73, 85, 99, and / or 111, the amino acid residue is Z4; (e) at positions 34 and / or 95, the amino acid residue is Z5; (f). 22nd, 25th, 29th, 43rd, 44th, 66th, 74th, 87th, 102nd, 108th, 116th, 122nd, 127th, 133th, 134th, 136th, and / Or at position 137, the amino acid residue is Z6; where Z1 is an amino acid selected from the group consisting of A, I, L, M, and V; Z2 is the group consisting of F, W, and Y. Z3 is an amino acid selected from the group consisting of N, Q, S, and T; Z4 is an amino acid selected from the group consisting of R, H, and K; Z5 is an amino acid selected from It is an amino acid selected from the group consisting of D and E; and Z6 is an amino acid selected from the group consisting of C, G, and P.

Some preferred GAT polypeptides of the invention are characterized as follows. If desired, at least 80% of the amino acid residues corresponding to the following positions in the polypeptide are aligned with the reference amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514. According to the following constraints: (a) at position 2, the amino acid residue is I or L; (b) at position 3, the amino acid residue is E or D; (c) at position 4, the amino acid residue is V, A or I; (d) at position 5 and amino acid residue is K, R or N; (e) at position 6 and amino acid residue is at position P or L; (f) at position 8 , Amino acid residues are N, S or T; (g) at position 10 and amino acid residues are E or G; (h) at position 11 and amino acid residues are D or E; (i) At position 12, the amino acid residue is T or A; (j) at position 14, the amino acid residue is E or K; (k) at position 15, the amino acid residue is I or L; (l) ) At position 17, the amino acid residue is H or Q; (m) at position 18, the amino acid residue is R, C or K; (n) at position 19, the amino acid residue is I or V. (O) At position 24, the amino acid residue is Q or R; (p) At position 26, the amino acid residue is L or I; (q) At position 27, the amino acid residue is at E or D There; (r) at position 28, the amino acid residue is A or V; (s) at position 30, the amino acid residue is K, M or R; (t) at position 31, the amino acid residue is Y Or F; (u) at position 32, the amino acid residue is E or G; (v) at position 33, the amino acid residue is at position T, A or S; (w) at position 35, the amino acid residue The group is L, S or M; at position (x) 37, the amino acid residue is R, G, E or Q; at position (y) 38, the amino acid residue is G or S; (z) ) At position 39, the amino acid residue is T, A or S; (aa) at position 40, the amino acid residue is F, L or S; (ab) at position 45, the amino acid residue is Y or F. Is;

Some preferred GAT polypeptides of the invention are characterized as follows. If desired, when aligned with a reference amino acid selected from the group consisting of SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514, at least 80% of the amino acid residues corresponding to the following positions in the polypeptide , (A) At positions 9, 76, 94 and 110, the amino acid residue is A; (b) At positions 29 and 108, the amino acid residue is C; (c) ) At position 34, the amino acid residue is D; (d) at position 95, the amino acid residue is E; (e) at position 56, the amino acid residue is F; (f) at position 43, 44 At positions 6, 66, 74, 87, 102, 116, 122, 127 and 136, the amino acid residue is G; (g) at 41, the amino acid residue is H; ( h) At position 7, the amino acid residue is I; (i) at position 85, the amino acid residue is K; (j) at position 20, 36, 42, 50, 72, 78, At positions 98 and 121, the amino acid residue is L; (k) at positions 1, 75 and 141, the amino acid residue is M; (l) at positions 23, 64 and 109, the amino acid remains. The group is N; (m) at positions 22, 25, 133, 134 and 137, the amino acid residue is P; (n) at position 71, the amino acid residue is Q; (o) ) At positions 16, 21, 73, 99 and 111, the amino acid residue is R; (p) at positions 55 and 88, the amino acid residue is S; (q) at position 77, The amino acid residue is T; (r) at position 107 and the amino acid residue is W; and (s) at positions 13, 46, 70, 117 and 118, the amino acid residue is Y.

Some preferred GAT polypeptides of the invention are characterized as follows. If desired, the amino acid residue in the polypeptide corresponding to position 28 is V or A when aligned with a reference amino acid selected from the group consisting of SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514. Valine in 28th place is generally reduced KMOn the other hand, alanine at this position generally increased kcatis connected with. Other preferred GAT polypeptides are I27 (ie, position 27 is I), M30, S35, R37, S39, G48, K49, N57, Q58, P62, Q65, Q67, K68, E83, S89, A96, E96, It is characterized by having R101, T112, A114, K119, K120, E128, V129, D131, T131, V134, R144, I145, or T146, or any combination thereof.

Some preferred GAT polypeptides of the invention include amino acid sequences selected from the group consisting of SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514.

The present invention further provides preferred GAT polypeptides characterized by a combination of amino acid residue position constraints described above.

In addition, the present invention provides GAT polynucleotides encoding the preferred GAT polypeptides described above, and complementary nucleotide sequences thereof.

Some aspects of the invention are particularly relevant to any subset of the above categories of GAT polypeptides having GAT activity as described herein. These GAT polypeptides are preferred for use, for example, as agents for conferring glyphosate resistance to plants. Examples of desired levels of GAT activity are described herein.

In one aspect, the GAT polypeptide comprises an amino acid sequence encoded by an isolated form of a naturally occurring nucleic acid isolated from a recombinant or natural source, such as a bacterial strain. Wild-type polynucleotides encoding such GAT polypeptides can be specifically screened by standard techniques known in the art. For example, the polypeptides defined by SEQ ID NOs: 6 to 10 were discovered by expression cloning of a sequence from the Bacillus strain exhibiting GAT activity, described in more detail below.

The present invention relates to nucleotide sequences encoding amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 5 and SEQ ID NOs: 11 to 262, their complements, and SEQ ID NOs: 6 to 10 and SEQ ID NOs: 263 to 514. Encoded by an isolated or recombinant polynucleotide, comprising a nucleotide sequence that hybridizes under stringent conditions over substantially the entire length of the nucleotide sequence selected from the group consisting of (including complements). It also includes isolated or recombinant polypeptides.

The present invention further comprises any polypeptide having GAT activity and encoded by a fragment of any GAT-encoding polynucleotide described herein.

The present invention also provides fragments of GAT polypeptides that can be spliced together to form functional GAT polypeptides. Splicing can be achieved in vitro or in vivo and can include cis splicing or trans splicing (ie, intramolecular or intermolecular splicing). This fragment may itself have GAT activity, but it is not required. For example, two or more segments of a GAT polypeptide can be split by intein, and removal of the intein sequence by cis splicing results in a functional GAT polypeptide. In another example, the encrypted GAT polypeptide can be expressed as two or more separate fragments, and transsplashing of these fragments results in the recovery of the functional GAT polypeptide. The various aspects of cis-splicing and trans-splicing, gene encryption, and the introduction of intervening sequences are described in more detail in US Patent Applications 09 / 517,933 and 09 / 710,686. Both are incorporated herein by reference in their entirety.

In general, the invention is encoded by any modified GAT polynucleotide induced by mutations, recombination recombination, and / or the diversity of polynucleotide sequences described herein. Contains polypeptides. In some aspects of the invention, GAT polypeptides are modified by single or multiple amino acid substitutions, deletions, insertions, or a combination of one or more of these modified forms. The substitution may be conservative or non-conservative, may or may not be modified in function, and new functionality may be added. Insertions and deletions can be important, for example, in the case of shortening significant fragments of the sequence, or in further fusion of the sequence to either the internal or N-terminal or C-terminal. In some embodiments of the invention, the GAT polypeptide comprises functional additions such as, for example, secretory signals, chloroplast transport peptides, purified tags, or any many functional functional groups apparent to those of skill in the art. It is part of the fusion protein. And that is described in more detail elsewhere herein.

The polypeptides of the invention may contain one or more modified amino acids. The presence of modified amino acids can be advantageous, for example, in (a) increasing the in vivo half-life of the polypeptide, (b) reducing or increasing the antigenicity of the polypeptide, and (c) increasing the storage stability of the polypeptide. .. For example, amino acids are co-translated or post-translated during recombinant production (eg, N-linked glycosylation at the NXS / T motif upon expression in mammalian cells), or by synthetic means. It will be modified.

Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, plenylated (eg, farnesylated, geranylgeranylated) amino acids, acetylated amino acids, acylated amino acids, PEGylated amino acids, biotinylated amino acids, carboxylated. Examples include amino acids and phosphorylated amino acids. Sufficient references to guide those skilled in the art of amino acid modification are extensive throughout the literature. An example protocol can be found in Walker (1998) Protein Protocols on CD-ROM Human Press, Towata, NJ.

Recombinant methods for producing and isolating the GAT polypeptide of the invention are described herein. In addition to recombinant production, polypeptides can be produced by direct peptide synthesis using solid phase technology. (Stewart et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J (1963) J. Am. Chem. Soc. 85: 2149-2154). Peptide synthesis can be performed using manual techniques or automatic control. For example, automated synthesis can be accomplished using the Applied Biosystems 431A peptide synthesizer (Perkin Elmer, Foster City, Calif.) According to the instructions provided by the manufacturer. For example, the partial sequences are chemically synthesized separately and can be linked by using a chemical method that provides the full length of the GAT polypeptide. Peptides can also be obtained from a variety of sources.

In another aspect of the invention, the GAT polypeptide of the invention is, for example, related to the activity, distribution, and expression of the GAT polypeptide in various tissues of a transgenic plant, eg, diagnostic use. It is used in the production of antibodies having.

The GAT homolog polypeptide for antibody induction does not require biological activity, but the polypeptide or oligopeptide must be antigenic. The peptide used to induce a specific antibody can have an amino acid sequence consisting of at least 10 amino acids, preferably at least 15 or 20 amino acids. A short sequence of GAT polypeptides can be fused with another protein, such as keyhole limpet hemocyanin, to produce antibodies against the chimeric molecule.

Methods for producing polyclonal and monoclonal antibodies are known to those of skill in the art and many antibodies are available. For example, Coligan (1991) Current Protocols in Immunology Wiley / Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th edition) Lange Medical Publications, Los Altos, CA, and the references cited therein; Goding (1986) Monoclonal Antibodies: Principles and Practice (2nd Edition) Academic Press, New York, NY; and Kohler and Milstein (1975) Nature. See 256: 495-497. Other techniques suitable for antibody preparation include sorting a library of recombinant antibodies in phage or similar vectors. See Huse et al. (1989) Science 246: 1275-1281; and Ward et al. (1989) Nature 341: 544-546. Specific monoclonal and polyclonal antibodies as well as antisera usually contain at least about 0.1 μM KDAnd preferably at least about 0.01 μM or more of KDAnd most representatively and most preferably K of 0.001 μM or more.DAnd combine.

For additional details on antibody production and manipulation techniques, see Borrebaeck (ed) (1995) Antibody Engineering, 2nd Edition Freeman and Company, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford, England (McCafferty), and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, NJ (paul) Can be found in.

(Variation of array) The GAT polypeptides of the invention include conservatively modified variations of the sequences disclosed herein as SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514. Such conservatively modified variations include substitutions, additions or deletions, which are either a single amino acid or a small proportion of amino acids in SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514. Modify, add, or delete (typically less than about 5%, more typically less than 4%, less than 2%, or less than 1%).

For example, a conservatively modified variation (eg, deletion) of the 146 amino acid polypeptide identified as SEQ ID NO: 6 herein is at least 140 amino acids long, preferably at least 141 amino acids, more preferably at least 144. It has amino acids, and even more preferably at least 146 amino acids, which correspond to deletions of less than about 5%, less than about 4%, less than about 2%, or less than about 1% of the polypeptide sequence.

Another example of a conservatively modified variation of the polypeptide identified as SEQ ID NO: 6 herein (eg, "conservatively substituted variation") is described in Table 2 (below). According to the six substitution groups, it contains "conservative substitutions" up to about 7 residues (ie, less than about 5%) of the 146 amino acid polypeptide.

The GAT polypeptide sequence homologs of the invention, including conservatively substituted sequences, can be used in GAT polypeptides, in GAT fusions having a signal sequence (eg, chloraplast targeting sequence), or for protein purification. May be present as part of a larger polypeptide sequence, such as that which occurs in the addition of one or more domains for (eg, polyhistidine segment, FLAG tag segment, etc.). In the latter case, the additional functional domain has little or no effect on the activity of the GAT portion of the protein, or this additional domain is produced by a post-synthesis processing step, such as by treatment with a protease. Can be removed.

(Definition of polypeptide by immunoreactivity) Since the polypeptides of the invention provide a novel class of enzymes with defined activity, i.e. acetylation of glyphosate, the polypeptides also provide, for example, novel structural features that can be recognized in immunological assays. To do. The production of antisera that specifically binds the polypeptides of the invention is one of the features of the invention, as well as the polypeptides bound by such antisera.

The present invention specifically binds or is specifically immunoreactive to an antibody or antiserum generated against an immunogen containing an amino acid sequence selected from one or more SEQ ID NOs: 6 to 10. Contains the GAT polypeptide that is. To eliminate cross-reactivity with other GAT homologs, this antibody or antiserum is a protein or peptide corresponding to the GenBank accession number available as of the filing date of the present application (they are by CAA70664, Z99109 and Y09476). Is removed by available related proteins, such as the proteins indicated by). In the case of the registration number corresponding to the nucleic acid, the polypeptide encoded by the nucleic acid is produced and used for the purpose of antibody / antiserum removal. Figure 3 tabulates the relative identity between the exemplary GAT polypeptide and the most closely related sequence available in Genbank, YitI. The function of the native YitI has not yet been elucidated, but the enzyme has been shown to have detectable GAT activity.

In one representative format, the immunoassay is one or more of SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514, or substantial subsequences thereof (ie, at least about 30% of the provided full-length sequence). Use polyclonal antisera produced for one or more polypeptides, including one or more sequences corresponding to. The entire set of possible polypeptide immunogens derived from SEQ ID NOs: 6-10 and SEQ ID NOs: 263-514 are collectively referred to below as "immunogenic polypeptides". The resulting antiserum was selected to have low cross-reactivity to other related sequences as needed, and any such cross-reactivity should be one prior to the use of the polyclonal antiserum in the immunoassay. It is removed by immunoadsorption using the above related sequences.

To produce antisera for use in immunoassays, one or more immunogenic polypeptides are produced and purified as described herein. For example, recombinant proteins can be produced in bacterial cell lines. Mouse intimacy (used in this assay because of the reproducibility of the results due to substantial mouse genetic identity) is combined with standard adjuvants such as Freund's adjuvant. Immunogenic proteins, and standard mouse immunoprotocols are used to immunize (antibody production that can be used to determine specific immunoreactivity, standard immunoassay formats and immunoassay conditions). See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York for a detailed description). Alternatively, one or more synthetic or recombinant polypeptides derived from the sequences disclosed herein are bound to a carrier protein and used as an immunogen.

The polyclonal sera are collected and titrated against the immunogenic polypeptide in an immunoassay, eg, a solid phase immunoassay with one or more immunogenic proteins immobilized on a solid support. Titer is 106The polyclonal antisera described above were selected, pooled, removed using a related polypeptide, eg, a polypeptide identified from GENBANK as shown, removed, pooled and titrated. To produce.

This removed, pooled and titrated polyclonal antiserum is tested for cross-reactivity to the associated polypeptide. Preferably, at least two immunogenic GATs are preferably used in this measurement together with at least two related polypeptides to identify antibodies to which the immunogenic protein specifically binds.

In this comparative assay, the binding conditions for differentiation are at least about 5 relative to the binding of the titrated polyclonal antiserum to the immunogenic GAT polypeptide compared to the binding to the associated polypeptide. Determined for removed and titrated polyclonal antisera, resulting in ~ 10-fold higher signal-to-noise ratio. That is, the stringency of the binding reaction is regulated by the addition of non-specific competitors such as albumin or skim milk powder, or by regulation of salt conditions, temperature and the like. These binding conditions will be used in the next assay to determine if the test polypeptide is specifically bound by the pooled and removed polyclonal antiserum. In particular, the test polypeptide exhibits a signal noise ratio that is at least 2-5 times higher than the control polypeptide under binding conditions for differentiation, and is at least about 1/2 compared to the immunogenic polypeptide. When the signal noise ratio of is shown, it shares substantial structural similarity with immunogenic polypeptides as compared to known GATs, and thus the test polypeptide is the polypeptide of the invention.

In another example, an immunoassay in a competitive binding format is used to detect the test polypeptide. For example, as shown, the cross-reactive antibody is removed from the pooled antiserum mixture by immunoadsorption with the control GAT polypeptide. The immunogenic polypeptide is then immobilized on a solid phase support, which is exposed to the removed and pooled antiserum. The test protein is added to the assay that competes for binding to the pooled and removed antiserum. The ability of this test protein to compete for binding to the pooled and removed antiserum compared to the fixed protein is compared to the ability of the immunogenic polypeptide added to this assay to compete for binding ( Immunogenic polypeptides effectively compete with fixed immunogenic polypeptides for binding to pooled antisera). The percent cross-reactivity of this test protein is calculated using standard formulas.

In a parallel assay, the ability of the control protein to compete for binding to the pooled and removed antiserum is determined, if necessary, compared to the ability of the immunogenic polypeptide to compete for binding to the antiserum. Again, the percent cross-reactivity to the control polypeptide is calculated using standard calculation methods. If the percent cross-reactivity is at least 5-10 fold higher than the test peptide, the test peptide is said to specifically bind to the pooled and removed antiserum.

In general, immunoadsorbed antisera and pooled antisera are used in competitive binding immunoassays such as those described herein to immunogenic polypeptides of any test polypeptide. Can be compared. To make this comparison, the two polypeptides were each assayed at a wide range of concentrations, and each required to inhibit 50% inhibition of the abstracted antiserum binding to the immobilized protein. The amount of polypeptide is determined using standard techniques. If the amount of test polypeptide required is less than twice the amount of immunogenic polypeptide required, then the test polypeptide is specific for the antibody produced against the immunogenic protein. The amount supplied, said to bind, is at least about 5-10 times higher than that of the control polypeptide.

As a final determination of specificity, the resulting immunogenic polypeptide is pooled until little or no binding of the abstracted and pooled antisera to the immunogenic polypeptide used in immunoadsorption is detected. The antiserum is completely adsorbed to the immunogenic polypeptide (rather than the control polypeptide), if desired. This fully immunoadsorbed antiserum is then tested for reactivity with the test polypeptide. When little or no reactivity is observed (ie, the ratio of the signal to the noise observed for the binding of the fully immunoadsorbed antiserum to the immunogenic polypeptide is doubled. If only), the test polypeptide is specifically bound by an immunogenic protein-induced antiserum.

(Glyphosate N-acetyltransferase polynucleotide) In one aspect, the invention is an isolated or recombinant polynucleotide referred to herein as "glyphosate N-acetyltransferase polynucleotide" or "GAT polynucleotide". Offering a new family of. The GAT polynucleotide sequence is characterized by its ability to encode a GAT polypeptide. Generally, the invention includes any nucleic acid sequence encoding any of the novel GAT polypeptides described herein. In some aspects of the invention, GAT polynucleotides encoding GAT polypeptides with GAT activity are preferred.

In one aspect, the GAT polynucleotide comprises a recombinant and isolated form of a naturally occurring nucleic acid isolated from an organism (eg, a bacterial strain). Exemplary GAT polynucleotides (eg, SEQ ID NOs: 1 to 5) were discovered by expression cloning of sequences derived from the Bacillus strain exhibiting GAT activity. Briefly, a collection of approximately 500 Bacillus and Pseudomonas strains was screened for their native ability to N-acetylate glyphosate. The strain was grown overnight in LB, harvested by centrifugation, permeated with dilute toluene, then washed and resuspended in a reaction mixture containing 5 mM glyphosate and 200 μM acetyl-CoA buffer. Cells were incubated in the reaction mixture for 1-48 hours, at which time equivalent volumes of methanol were added to the reaction. Cells were then pelleted by centrifugation and the supernatant was filtered prior to analysis by parent ion mode mass spectrometry. The product of the reaction was clearly identified as N-acetylglyphosate by comparing the mass spectrometric profile of the reaction mixture with the standard for N-acetylglyphosate as shown in FIG. Product detection relied on encapsulation of both substrates (acetyl-CoA and glyphosate) and was extinguished by thermal denaturation of bacterial cells.

Individual GAT polynucleotides were then cloned from strains identified by functional screening. Genomic DNA was prepared and partially digested with the Sau3A1 enzyme. A fragment of about 4 Kb was cloned into an E. coli expression vector and transformed into an electrically competent E. coli. Individual clones exhibiting GAT activity were identified by post-reaction mass spectrometric methods as previously described, except that toluene washing was replaced by permeabilization with PMBS. Genomic fragments were sequenced and putative GAT polypeptides encoding open reading frames were identified. Identification of the GAT gene was confirmed by expression of an open reading frame in E. coli and detection of high levels of N-acetylglyphosate produced from the reaction mixture.

In another aspect of the invention, GAT polynucleotides are recombinant and / or mutated by diversification (eg, one or more naturally occurring isolated GAT polynucleotides or recombinant GAT polynucleotides). Produced (by). As described in more detail elsewhere herein, superior functional properties (eg, increased catalytic function, increased stability over GAT polynucleotides used as substrates or parents in diversification processes). It is often possible to produce diversified GAT polynucleotides that encode GAT polypeptides with high expression levels).

The polynucleotides of the invention have a variety of uses, eg: recombinant production (ie, expression) of the GAT polypeptide of the invention; as a transgene (eg, to confer herbicide resistance to a transgenic plant). ); As a selective marker for transformation and plasmid maintenance; as an immunogen; as a diagnostic probe for the presence of complementary or partially complementary nucleic acids (included for the detection of native GAT-encoding nucleic acids) As a substrate for further diversity production (eg, recombination or mutation reactions to produce new GAT homologs and / or improved GAT homologs, etc.).

It is important to note that certain substantial and reliable utility of GAT polynucleotides does not require a polynucleotide encoding a polypeptide having substantial GAT activity. For example, GAT polynucleotides that do not encode active enzymes have desirable functional properties (eg, high kcat or kcat / Km, low Km, high stability to heat or other environmental factors, high transcription or translation rate, protein solubility. A valuable source of parent polynucleotide for use in diversified procedures to reach GAT polynucleotide variants or non-GAT polynucleotides that have resistance to cleavage, reduced antigenicity, etc.) obtain. For example, a nucleotide sequence encoding a protease variant with little or no detected activity is used as the parent polynucleotide in a DNA shuffling experiment to produce progeny encoding a highly active protease (Ness et al., (1999). ) Nature Biotechnology 17: 893-96).

Polynucleotide sequences produced by diversity generation methods or recombination recombination (RSR]) methods (eg, DNA shuffling) are characteristic of the present invention, using the nucleic acids described herein. Mutation and recombination methods are characteristic of the present invention. For example, one method of the present invention combines one or more nucleotide sequences of the present invention described above and below with one or more additional sequences. Includes the step of recombination. This recombination step is performed in vivo, exovivo, incilico or in vitro as needed. The diversity generation or recombination sequence recombination described above is a recombinant modified GAT. Produces at least one library of polynucleotides. Polypeptides encoded by members of this library are included in the invention.

Also as oligonucleotides are the use of polynucleotides referred to herein (typically having at least 12 bases, preferably at least 15 bases, more preferably at least 20, 30, or 50 or more bases). Also contemplated, it hybridizes to the GAT polynucleotide sequence under stringent or highly stringent conditions. Polynucleotides can be used as probes, primers, senses, antisense factors, etc., according to the methods described herein.

According to the present invention, GAT polynucleotides, including GAT polypeptides, fragments of GAT polypeptides, associated fusion proteins, or nucleotide sequences encoding functional equivalents thereof, are suitable hosts such as bacterial or plant cells. It is used in recombinant DNA molecules that direct the expression of GAT polypeptides in cells. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences encoding substantially the same amino acid sequence or functionally equivalent amino acid sequences can also be used to clone and express GAT polynucleotides. ..

The present invention provides GAT polynucleotides that subsequently encode transcripts and / or translations spliced to produce functional GAT polypeptides. Splicing can be accomplished in vitro or in vivo and can include cis splicing or trans splicing. The substrate for splicing can be a polynucleotide (eg, an RNA transcript) or a polypeptide. An example of cis-splicing a polynucleotide is when the intron inserted into the coding sequence is removed and two adjacent exon regions are spliced to produce the GAT polypeptide coding sequence. An example of trans-splicing is when a GAT polynucleotide is transcribed separately and then spliced and encrypted by splitting the coding sequence into two or more fragments that can form a full-length GAT coding sequence. .. The use of splicing enhancer sequences, which can be introduced into the constructs of the present invention, can facilitate splicing of either cis or trans. Sith splicing and trans splicing of polypeptides are described in more detail elsewhere herein. A more detailed description of cis-splicing and trans-splicing can be found in US Patent Applications 09 / 517,933 and 09 / 710,686.

Thus, some GAT polynucleotides do not directly encode a full-length GAT polypeptide, but rather a fragment of the GAT polypeptide. These GAT polynucleotides can be used to express functional GAT polypeptides through mechanisms involved in splicing. Here, splicing can occur at the level of polynucleotides (eg, introns / exons) and / or polypeptides (eg, intein / extein). In experiments that allow the splicing process to produce functional products, this is, for example, the regulation of GAT activity, since when all required fragments are expressed, only the functional GAT polypeptide is expressed. Can be useful for. In another example, the introduction of one or more inserts into a GAT polynucleotide may facilitate recombination with a polynucleotide of low homology; the use of an intron or intein for the insert sequence removes the intervening sequence. Facilitates and thereby restores the functionality of the encoded variant.

As will be appreciated by those skilled in the art, it may be advantageous to modify the coding sequence to enhance its expression in a particular host. Although 64 possible codons are degenerate in its genetic code, most organisms preferentially use some of these codons. The most commonly used codons in one species are called optimal codons, and less commonly used codons are classified as rare or underused codons (eg, Zhang SP et al., (1991) Gene 105: See 61-72). Codons are replaced to respond to the host's preferred codon usage, and this process is sometimes referred to as "codon optimization" or "control of species codon tendencies."

An optimized coding sequence containing codons preferred by a particular prokaryotic or eukaryotic host (see also Murray, E. et al., (1989) Nuc. Acids Res. 17: 477-508) is, for example, non-. It can be prepared to produce recombinant RNA transcripts with desired properties such as increased translation rate or longer half-life compared to transcripts produced from optimized sequences. The translation stop codon can also be modified to reflect the host's priority. For example, the preferred stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The preferred stop codon for monocotyledonous plants is UGA, whereas insects and E. coli prefer to use UAA as the stop codon (Dalphin ME et al., (1996) Nuc. Acids Res. 24: 216- 218). Methodologies for optimizing nucleotide sequences for expression in plants are provided, for example, in US Pat. No. 6,015,891 and references cited therein.

One embodiment of the invention comprises a GAT polynucleotide having an optimal codon for expression in a related host (eg, a transgenic plant host). This is especially desirable when introducing a GAT polynucleotide of bacterial origin into a transgenic plant, for example, to confer glyphosate resistance on the plant.

The polynucleotide sequences of the invention can be designed to alter GAT polynucleotides for a variety of reasons, including but not limited to: cloning and processing of gene products. And / or modifications that modify expression. For example, modification introduces techniques well known to those of skill in the art (eg, site-specific mutagenesis, insertion of new restriction sites, modification of glycosylation patterns, changing codon priorities, splicing sites. Can be introduced using things like that).

As described in more detail herein, the polynucleotides of the invention are a sequence encoding a novel GAT polypeptide, a complementary sequence to this coding sequence, and a novel fragment of the coding sequence, and Includes its complement. These polynucleotides can be in RNA or DNA form and include mRNA, cRNA, synthetic RNA and synthetic DNA, genomic DNA and cDNA. This polynucleotide can be double-stranded or single-stranded, and in the case of single-stranded, it can be a coding strand or a non-coding (antisense, complementary) strand. This polynucleotide optionally contains the coding sequence of the GAT polypeptide as follows: (i) Isolation and (ii) Further coding sequences to encode, for example, fusion proteins, preproteins, preproproteins, etc. In combination with (iii) non-coding sequences such as introns or inteins, promoters, enhancers, terminator elements, or 5'and / or 3'untranslated regions useful for expression of coding sequences in suitable hosts. In combination with a regulatory element, and / or (iv) a vector or host environment in which the GAT polynucleotide is a heterologous gene. Sequences can also be found in combination with representative composition formulations of nucleic acids, including in the presence of carriers, buffers, adjuvants, excipients and the like.

The polynucleotides and oligonucleotides of the present invention can be prepared by standard solid phase methods according to known synthetic methods. Typically, fragments up to about 100 bases are synthesized individually and then bound to form substantially any desired sequence (eg, enzymatic or chemical binding, or polymerase). By mediation method). For example, the polynucleotides and oligonucleotides of the present invention are described, for example, in the classical phosphoramidite method described by Beaucage et al., (1981) Tetrahedron Letters 22: 1859-69, or Matthes et al., (1984) EMBO J. 3:801. It can be prepared by chemical synthesis using the method described by -05 (eg, methods typically performed by automated synthetic methods). According to the phosphoramidite method, oligonucleotides are synthesized, for example, in an automated DNA synthesizer, purified, annealed, ligated, and cloned into the appropriate vector.

In addition, virtually any nucleic acid is available at The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (http://www.genco.com), ExpressGen Inc. (www.expressgen.com). , Operon Technologies Inc. (Alameda, CA) and many others can be specially ordered from any variety of commercial sources. Similarly, peptides and antibodies include PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc. (http://www.htibio.com), BMA Biomedicals Ltd (UK), Bio.Synthesis, Inc. and It can be specially ordered from any variety of sources such as many others.

Polynucleotides can also be synthesized by well-known techniques as described in the technical literature. See, for example, Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47: 411-418 (1982), and Adams et al., J. Am. Chem. Soc. 105: 661 (1983). Double-stranded DNA fragments are then complemented by synthesizing complementary strands and annealing these strands together under the appropriate conditions, or by using DNA polymerase with the appropriate primer sequence. Can be obtained by adding to either.

Common texts describing useful molecular biology techniques herein, including mutagenesis, include: Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, CA ("Berger"); Sambook et al., Molecular Cloning-A Laboratory Manual (2nd Edition), Volumes 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ( "Sambrook"); and Current Protocols in Molecular Biology, FM Ausubel et al., Eds., Current Protocols, Greene Publishing Associates, Inc. and John Merger with Wiley & Sons, Inc. (complemented throughout 2000) ("Ausubel"). Sufficient technique to direct those skilled in the art through in vitro augmentation methods including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ replica-se amplification and other RNA polymerase mediated techniques (eg NASBA). Examples of are found below: Berger, Sambrook, and Ausubel and Mullis et al., (1987) US Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al., Ed.) Academic Press Inc. San Diego, CA (1990); Arnheim & Levinson (October 1, 1990) Chemical and Engineering News 36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al., (1989) Proc.Natl.Acad.Sci .USA 86: 1173; Guatelli et al., (1990) Proc.Natl.Acad.Sci.USA 87: 1874; Lomell et al., (1989) J.Clin.Chem.35:1826; Landegren et al., (1988) Science 241: 1077- 1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace, (1989) Gene 4: 560; Barringer et al., (1990) Gene 89: 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564. An improved method for cloning in vitro amplified nucleic acids is described in Wallace et al., US Pat. No. 5,426,039. Improvements in the amplification of large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and references therein, where PCR amplicons up to 40 kb are produced. One of skill in the art recognizes that substantially any RNA is converted to double-stranded DNA suitable for restriction digestion, PCR elongation and sequencing using reverse transcriptase and polymerase. See Ausbel, Sambrook and Berger (all above).

(Array variation) Due to the degeneracy of the genetic code, a large number of nucleotide sequences encoding the GAT polypeptides of the invention can be generated, some of which are substantially identical to the nucleic acid sequences expressly disclosed herein. Having sex is recognized by those skilled in the art.

<tables num="1"><img file="JP2010142234A_D0001.tif" /></tables> For example, inspection of the codon table (Table 1) shows that all of the codons AGA, AGG, CGA, CGC, CGG and CGU encode the amino acid arginine. Thus, at all positions of the nucleic acid of the invention in which arginine is identified by a codon, the codon can be converted to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that the U in the RNA sequence corresponds to the T in the DNA sequence.

Using the nucleic acid sequence corresponding to nucleotides 1-15 of SEQ ID NO: 1 (ATG ATT GAA GTC AAA) as an example, a silent variation of this sequence comprises AGT ATC GAG GTG AAG, both of which are SEQ ID NOs. It encodes the amino acid sequence MIEVK corresponding to amino acids 1 to 5 of 6.

Such a "silent variation" is a type of "conservative modification variation" and is discussed below. Those skilled in the art will recognize that each codon of nucleic acid (except AUG, which is usually the only codon for methionine) can be modified by standard techniques to encode a functionally identical polypeptide. Therefore, each of the silent variations of the nucleic acid encoding the polypeptide is latent in any of the described sequences. The present invention provides each and all possible variations of the nucleic acid sequences encoding the polypeptides of the invention, which can be made by selecting combinations based on possible codon selection. These combinations are made according to the standard triplet genetic code (eg, as shown in Table 1) when applied to the nucleic acid sequences encoding the GAT homolog polypeptides of the invention. All such variations of all nucleic acids herein are specifically provided and described in combination with the genetic code, taking into account the sequence. Any variant can be produced as described herein.

A group of two or more different codons, all encoding the same amino acid, are referred to in the favorite book as "synonymous codons" when translated in the same context. As described herein, in some aspects of the invention, GAT polynucleotides are designed for optimized codon use in desirable host organisms, such as plant hosts. The term "optimized" or "optimal" does not mean that it is limited to the best possible combination of codons, but simply the coding sequence as a whole is with the precursor polynucleotide from which it is derived. By comparison, it shows an improved use of codons. Thus, in one aspect, the invention refers to at least one parent codon in a nucleotide sequence by a synonymous codon that is preferentially used in a desired host organism, eg, a plant, as compared to the parent codon. Provided is a method for producing a GAT polynucleotide variant by substitution.

A "conservative modification variation" of a particular nucleic acid sequence, or simply a "conservative variation," is a nucleic acid that encodes the same amino acid sequence or a substantially identical amino acid sequence, or if the nucleic acid does not encode an amino acid sequence. Refers to substantially the same sequence. Those skilled in the art may modify or add a single amino acid or a low% amino acid (typically less than 5%, more typically less than 4%, 2% or less than 1%, or less) within the encoded sequence. Or that individual substitutions, deletions or additions that are deleted are "conservative modification variations", and that this modification results in amino acid deletions, amino acid additions, or amino acid substitutions with chemically similar amino acids. recognize.

Conservative substitution tables that provide functionally similar amino acids are well known in the art. Table 2 shows six groups containing amino acids that are "conservative substitutions" with each other.

<tables num="2"><img file="JP2010142234A_D0002.tif" /></tables> Therefore, the "conservative substitution variation" of the listed polypeptides of the invention is a low percentage (typically less than 5%) of the amino acids in the polypeptide sequence by the conservatively selected amino acids of the same conservative substitution group. , More typically less than 2% and often less than 1%).

For example, the conservative substitution variation of the polypeptide identified herein as SEQ ID NO: 6 is defined above up to 7 residues (ie, 5% amino acids) within a 146 amino acid polypeptide. Includes "conservative replacement" according to the group.

In a further example, if the four conservative substitutions are localized to the region corresponding to amino acids 21-30 of SEQ ID NO: 6, an example of a conservative substitution variation for this region is RPN QPL EAC M,KPQ QPV ESCM andKPN NPL DAC V Follow the conservative substitutions listed in Table 2 (in the above example, the conservative substitutions are underlined). The list of protein sequences herein, together with the above substitution table, provides a clear table of all conservative substitution proteins.

Finally, the addition of sequences that do not alter the encoded activity of the nucleic acid molecule (eg, the addition of non-functional or non-coding sequences) is a conservative variation of the basic nucleic acid.

Those skilled in the art will recognize that many conservative variations of the disclosed nucleic acid constructs result in functionally identical constructs. For example, as described above, due to the degeneracy of the genetic code, "silent substitutions" (ie, substitutions of nucleic acid sequences that do not result in alterations in the encoded polypeptide) are all encoding amino acids. Is a meaningful feature of the nucleic acid sequence of. Similarly, "conservative amino acid substitutions" of one or several amino acids within an amino acid sequence are easily replaced by different amino acids with very similar properties and are also very similar to the disclosed constructs. Be identified. Conservative variations of each such disclosed sequence are characteristic of the present invention.

Non-conservative modifications of a particular nucleic acid are modifications that replace any amino acid that is not characterized as a conservative substitution. For example, any substitutions that cross the boundaries of the six groups are shown in Table 2. These are basic or acidic amino acid substitutions for neutral amino acids (eg, Asp, Glu, Asn or Gln for Val, Ile, Leu or Met), aromatic amino acid substitutions for basic or acidic amino acids (eg, for example). Includes Phe, Tyr or Trp) for Asp, Asn, Glu or Gln or any other substitution that does not replace an amino acid with a similar amino acid.

(Nucleic acid hybridization) Nucleic acids "hybridize" when they associate (typically in solution). Nucleic acids hybridize due to various well-characterized physicochemical forces such as hydrogen bonding, solvent exclusion, base stacking, and the like. Extensive guidelines for nucleic acid hybridization can be found below in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and The strategy of nucleic acid probes assays, (Elsevier, New York), and Ausubel, Kamiide, Hames and Higgins (1995) Gene Probes 1, IRL Press, Oxford University. Press, Oxford, England (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes 2, IRL Press, Oxford University Press, Oxford, England (Hames and Higgins 2) synthesize and label oligonucleotide-containing DNA and RNA. , Provides details regarding detection and quantification.

In the context of nucleic acid hybridization experiments such as Southern Hybridization and Northern Hybridization, "stringent hybridization wash conditions" are sequence-dependent and differ under different environmental parameters. Extensive guidelines for nucleic acid hybridization can be found in Tijssen (1993), supra, and Hames and Higgins 1 and Hames and Higgins 2, supra.

For the purposes of the present invention, generally "highly stringent" hybridization and washing conditions are the melting points (T) for a particular sequence at defined ionic strength and pH.m) Is chosen to be less than or equal to approximately 5 ° C (as noted below, highly stringent conditions can also be referred to in comparative terms). TmIs the temperature at which 50% of the test sequence hybridizes to a perfectly matched probe (under specified ionic strength and pH). A very stringent condition is the T for a particular probemIs selected to be equal to.

Nucleic acid duplex TmIndicates the temperature at which the duplex is denatured by 50% under certain conditions, and represents a direct measure of the stability of the nucleic acid hybrid. Therefore, TmCorresponds to the temperature corresponding to the midpoint of the transition from helix to random coil; TmDepends on length, nucleotide composition and ionic strength for long stretches of nucleotides.

After hybridization, the non-hybridized nucleic acid material can be removed by a series of washes, and the stringency of the washes can be adjusted depending on the desired result. Low stringency wash conditions (eg, conditions with higher salt concentrations and lower temperatures) increase sensitivity but can result in non-specific hybridization signals and high background signals. Higher stringency conditions (eg, lower salt concentrations and conditions with higher temperatures, closer to the hybridization temperature) reduce the background signal, leaving typically only specific signals. To do. See Rapley, R. and Walker, JM, Molecular Biomethods Handbook (Humana Press, Inc. 1998) (hereinafter "Rapley and Walker" herein), which is incorporated herein by reference. As a reference, the whole is used for all purposes.

DNA-DNA double strand TmCan be estimated using Equation 1 such as: Tm(° C) = 81.5 ° C + 16.6 (log10M) + 0.41 (% G + C) -0.72 (% f) -500 / n, Where M is the molar concentration of monovalent cations (usually Na +), (% G + C) is the percentage of guanosine (G) and cytosine (C) nucleotides, (% f) is the percentage of formamide, and n is The number of nucleotide bases (ie, length) of the hybrid. Rapley and Walker, see above.

RNA-DNA double strand TmCan be estimated by using Equation 2 as follows: Tm(° C) = 79.8 ° C + 18.5 (log10M) +0.58 (% G + C) -11.8 (% G + C)2-0.56 (% f) -820 / n, Where M is the molar concentration of monovalent cations (usually Na +), (% G + C) is the percentage of guanosine (G) and cytosine (C) nucleotides, (% f) is the percentage of formamide, and n. Is the number of nucleotide bases (ie, length) of the hybrid. Same as above.

Formulas 1 and 2 are typically accurate only for hybrid duplexes longer than about 100-200 nucleotides. Same as above.

T of nucleic acid sequence shorter than 50 nucleotidesmCan be calculated as: Tm(° C) = 4 (G + C) + 2 (A + T), Where A (adenine), C, T (thymine), and G are the number of corresponding nucleotides.

An example of stringent hybridization conditions for hybridization of complementary nucleic acids with more than 100 complementary residues on the filter in Southern or Northern blots is supplemented with 1 mg heparin at 42 ° C. 50% formalin and hybridization is to be performed overnight. An example of stringent wash conditions is 0.2 x SSC wash at 65 ° C for 15 minutes (see Sambrook et al., Supra, for a description of SSC buffer). Often, high stringency lavages outperform low stringency lavages to eliminate background probe signals. An example of a low stringency wash is 2 x SSC at 40 ° C for 15 minutes.

In general, a noise ratio signal that is 2.5 to 5 times (or even higher) than the noise ratio signal found in unrelated probes in a particular hybridization assay indicates detection of specific hybridization. Detection of at least stringent hybridization between two sequences under the context of the invention is, for example, the relatively strong structural similarity or homology to the nucleic acids of the invention provided in the sequence listings herein. Show sex.

As noted, the "highly stringent" condition is the thermal melting point (T) for a particular sequence at defined ionic strength and pH.m) Is selected so that it is about 5 ° C or less. Target sequences that are close to or identical to the nucleotide sequence of interest (eg, "probe") can be identified under highly stringent conditions. Low stringent conditions are appropriate for less complementary sequences. See, for example, Rapley and Walker above.

Comparative hybridization can be used to identify the nucleic acids of the invention, and this method of comparative hybridization is the preferred method of distinguishing the nucleic acids of the invention. Detection of highly stringent hybridization between two nucleotide sequences under the context of the present invention is, for example, relatively strong structural similarity / homology to the nucleic acids provided in the sequence listings herein. Show sex. Highly stringent hybridization between two nucleotide sequences has a higher degree of structural similarity or homology, nucleotide base composition similarity or homology, than is detected by stringent hybridization conditions. Shows similarity or homology in arrangement or order. In particular, the detection of highly stringent hybridization under the context of the present invention is, for example, strong structural similarity or structural homology (eg, nucleic acid structure, bases) to the nucleic acids provided in the sequence listings herein. Composition, base arrangement or base order) is shown. For example, it is desired to identify test nucleic acids that hybridize to the exemplary nucleic acids herein under stringent conditions.

Thus, one measure of stringent hybridization is the enumerated nucleic acids (eg, the nucleic acid sequences of SEQ ID NOs: 1 to 5, and the nucleic acid sequences of SEQ ID NOs: 11 to 262, and their complementary polys. Under highly stringent conditions (or very stringent conditions, or ultra-high stringency hybridization conditions, or ultra-ultra-high stringency hybridization conditions) for one of the nucleotide sequences). It is the ability to hybridize with. Stringent hybridization (and highly stringent hybridization, ultra-highly stringent hybridization, or ultra-ultra-highly stringent hybridization) and stringent wash conditions are for any test nucleic acid. It can be easily determined empirically. For example, in determining highly stringent hybridization conditions and highly stringent wash conditions, the hybridization and wash conditions are until the combination of selected criteria is met (eg, in hybridization or wash). It is gradually intensified by increasing temperature, decreasing salt concentration, increasing detergent concentration and / or increasing concentration of organic solvent (eg, formalin). For example, the hybridization and washing conditions are such that a probe containing one or more nucleic acid sequences selected from SEQ ID NOs: 1 to 5 and SEQ ID NOs: 11 to 262 and their complementary polynucleotide sequences is complete. To a complementary target that matches (again, a nucleic acid containing one or more nucleic acid sequences selected from SEQ ID NOs: 1 to 5 and SEQ ID NOs: 11 to 262 and their complementary polynucleotide sequences). In contrast, at least about 2. of the noise ratio signal observed in probe hybridization to non-matching targets. It is gradually strengthened until it is coupled with a noise ratio signal that is 5 times stronger, and in some cases about 5 times stronger. In this case, the non-matching target is a public database (eg GenBank) at the time of filing this application.TM) Corresponds to the nucleic acid (other than the nucleic acid in the attached sequence listing). One of ordinary skill in the art can identify such sequences in GenBank. Examples include registration numbers Z99109 and Y09476. One of ordinary skill in the art can identify additional such sequences, eg, in GenBank.

If the test nucleic acid hybridizes to the probe at least 1/2 the rate of the case against a perfectly matched complementary target, i.e., the perfectly matched probe is any non-registration number Z99109 and registration number Y09476. Conditions that bind to a perfectly matched complementary target with a noise ratio signal that is at least about 2 to 10 times, and in some cases 20 times, 50 times or more, the noise ratio signal observed in hybridization to a matching polynucleotide. A noise ratio signal that is at least half the height of hybridization of the probe to the target below is said to specifically hybridize to the probe nucleic acid.

Ultra-high stringency hybridization and washing conditions are any non-matching noise ratio signals for probe binding to complementary target nucleic acids for which the hybridization and washing conditions stringency are perfectly matched. It is enhanced to at least 10-fold higher than the noise ratio signal observed in probe hybridization to the target nucleic acids (Genbank Registration No. Z99109 and Registration No. Y09476). Target nucleic acids that hybridize to the probe under such conditions with a noise ratio signal of at least 1/2 of the noise ratio signal to the perfectly matched complementary target nucleic acid are probed under ultra-high stringency conditions. Is stated to combine with.

Similarly, even higher levels of stringency can be determined by progressively increasing hybridization and / or wash conditions for the relevant hybridization assay. For example, the noise ratio signal for probe binding to an exact matching complementary target nucleic acid is at least 10 of the noise ratio signals observed in hybridization to any non-matching target nucleic acid Genbank registration number Z99109 and registration number Y09476. A level of stringency in which the stringency of hybridization and washing conditions is enhanced until the altitude is fold, 20 fold, 50 fold, 100 fold, or 500 fold or higher. Target nucleic acids that hybridize to the probe under such conditions, with a noise ratio of at least 1/2 of the perfectly matched complementary target nucleic acid, are subject to ultra-ultra-high stringency conditions to the probe. Is stated to combine.

A target nucleic acid that hybridizes to the nucleic acids represented by SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 11 to SEQ ID NO: 262 under high, ultra-high, or ultra-ultra-high stringency conditions is one of the present inventions. It is an aspect. Examples of such nucleic acids include one or several silent nucleic acid substitutions or conservative nucleic acid substitutions compared to a given nucleic acid sequence.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if their encoding polypeptides are substantially identical. This happens, for example, with SEQ ID NO: 6 to SEQ ID NO: 10 and SEQ ID NO: 263 to SEQ ID NO: 514 subtracted using a polypeptide encoded by a known nucleotide sequence (including GenBank registration number CAA70664). If a copy of the nucleic acid is produced using the maximum codon degeneracy allowed by the gene code of, or deducted using a polypeptide encoded by a known nucleotide sequence (including GenBank registration number CAA70664). In addition, when anti-serum is prepared for one or more of SEQ ID NO: 6 to SEQ ID NO: 10 and SEQ ID NO: 263 to SEQ ID NO: 514. Further details on the immunological identity of the polypeptides of the invention are found below. In addition, TMAC1 hybridization procedures known to those of skill in the art can be used to distinguish between duplexes having sequences of less than about 100 nucleotides. For example, Sorg, U. et al., 1 Nucleic Acids See Res. (Sept. 11, 1991) 19 (17), which is incorporated herein by reference in its entirety for all purposes.

As one aspect, the present invention provides nucleic acids containing unique partial sequences in nucleic acids selected from SEQ ID NOs: 1 to 5 and SEQ ID NOs: 11 to 262. The unique subsequence is unique compared to the nucleic acid corresponding to either GenBank registration number Z99109 or registration number Y09476. Such a unique subsequence is relative to the complete set of nucleic acids represented by GenBank registration number Z99109, registration number Y09476 or other related sequences available in the public database as of the filing date of the present application. It can be determined by aligning any of the sequences of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 11 to SEQ ID NO: 262. Alignment can be performed using the BLAST algorithm set to the default parameters. Any unique partial sequence is useful, for example, as a probe for identifying the nucleic acids of the invention.

Similarly, the present invention includes polypeptides comprising a unique partial sequence in a polypeptide selected from SEQ ID NO: 6 to SEQ ID NO: 10 and SEQ ID NO: 263 to SEQ ID NO: 514. Here, the unique partial sequence is unique compared to the polypeptide corresponding to GenBank registration number CAA70664. Again, here the polypeptide is aligned with respect to the sequence represented by registration number CAA70664. If the sequence corresponds to an untranslated sequence such as a pseudogene, it should be noted that the corresponding polypeptide is simply made by incilico translation of the nucleic acid sequence into the amino acid sequence, where the reading frame is It is selected to correspond to the reading frame of the homologous GAT polynucleotide.

The present invention also provides stringent conditions for a unique coding oligonucleotide that encodes a unique partial sequence in a polypeptide selected from SEQ ID NO: 6 to SEQ ID NO: 10 and SEQ ID NO: 263 to SEQ ID NO: 514. Provides a target nucleic acid to hybridize with, where unique subsequences are unique compared to the polypeptide corresponding to any control polypeptide. The unique sequence is determined as described above.

In one example, stringent conditions are such that an oligonucleotide that is completely complementary to the encoding oligonucleotide is more than a hybridization of an oligonucleotide that is completely complementary to the control nucleic acid corresponding to any control polypeptide. It is selected to hybridize to the encoding oligonucleotide with a noise ratio signal that is at least about 2.5 to 10 times higher, preferably at least about 5 to 10 times higher. Conditions may be selected such that higher noise ratio signals (eg, about 15-fold, about 20-fold, about 30-fold, about 50-fold, or more) are seen in the particular assay used. In this example, the target nucleic acid hybridizes to a unique coding oligonucleotide with a noise ratio signal that is at least 2-fold higher than the hybridization of the control nucleic acid to the encoding oligonucleotide. Again, higher noise ratio signals (eg, about 2.5x, about 5x, about 10x, about 20x, about 30x, about 50x or more) can be selected. The particular signal depends on the label used in the relevant assay (eg, fluorescent label, colorimetric label, radioactive label, etc.).

(Vector, promoter and expression system) The present invention also includes recombinant constructs comprising one or more nucleic acid sequences broadly described above. The construct includes a vector in which the nucleic acid sequence of the present invention is integrated in the forward or reverse direction (for example, plasmid, cosmid, phage, virus, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), etc.). In a preferred aspect of this embodiment, the construct further comprises a regulatory sequence operably linked to the sequence (eg, including a promoter). Many suitable vectors and promoters are known to those of skill in the art and are commercially available.

Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, as general texts describing useful molecular biology techniques herein, including the use of vectors, promoters and many other related subjects. Volume 152, Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Edition), Volumes 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook"), and Current Protocols in Molecular Biology, edited by FM Ausubel et al., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & A joint venture of Sons, Inc. (supplemented until 1999) (Ausubel) can be mentioned. In vitro, including, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ replicase amplification, and other RNA polymerase-mediated techniques (eg, NASBA) for producing the homologous nucleic acids of the invention. Examples of protocols sufficient to direct those skilled in the art via amplification methods are Berger, Sambrook, and Ausubel, and Mullis et al. (1987) US Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al.). Ed.) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990) C & EN 36-47; The Journal Of NIH Research (1991) 3,81-94; ( Kwoh et al. (1989) Proc.Natl.Acad.Sci.USA 86,1173; Guatelli et al. (1990) Proc.Natl.Acad.Sci.USA 87,1874; Lomell et al. (1989) J.Clin.Chem 35,1826; Landegren et al. (1988) Science 241,1077-1080; Van Brunt (1990) Biotechnology 8,291-294; Wu and Wallace, (1989) Gene 4,560; Barringer et al. (1990) Gene 89,117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564. An improved method for cloning in vitro amplified nucleic acids is described in Wallace et al., US Pat. No. 5,426,039. An improved method for amplifying nucleic acids on a large scale by PCR is described in Cheng et al. (1994) Nature. Summarized in 369: 684-685 and the references cited therein, where 40 kb of PCR amplicon is produced. Those skilled in the art will appreciate that reverse transcriptases and polymerases can be used to convert virtually any RNA into double-stranded DNA suitable for restriction digestion, PCR elongation and sequencing. See, for example, Ausubel, Sambrook, and Berger (all ibid.).

The invention also transduces (transforms or transfects) the vectors of the invention (eg, the cloning vectors of the invention, or the expression vectors of the invention), engineered host cells, and recombinant techniques. Related to the production of the polypeptide of the invention according to. The vector can be, for example, a plasmid, viral particles, phages and the like. Manipulated host cells can be cultured in conventional nutrient media modified to be suitable for promoter activation, transformant selection, or amplification of the GAT homolog gene. Culture conditions (eg, temperature, pH, etc.) are those conventionally used in host cells selected for expression, and are apparent to those of skill in the art and are described herein (eg, reference). , Sambrook, Ausubel and Berger, and, for example, Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3rd Edition, Wiley-Liss, New York, and references cited therein). It is in.

The GAT polypeptides of the invention can be produced in non-animal cells (eg, plants, yeasts, fungi, bacteria, etc.). For more information on non-animal cell culture, in addition to Sambrook, Berger and Ausubel, Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995). ) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York), and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL ..

The polynucleotides of the invention can be incorporated into any of a variety of expression vectors suitable for expression of the polypeptide. Suitable vectors include chromosomal, non-chromosomal and synthetic DNA sequences (eg, SV40 derivatives; bacterial plasmids, phage DNA, baculovirus, yeast plasmids, as well as plasmid and phage DNA, viral DNA (eg, vaccinia, adenovirus, poultry). Vectors derived from a combination of viruses, pseudorabies, adenoviruses, adeno-associated viruses, retroviruses and many other viruses). All vectors that introduce plasmids into cells, and replication is desired. If so, all vectors that are replicable and viable in the relevant host can be used.

When incorporated into an expression vector, the polynucleotides of the invention are operably linked to the appropriate transcriptional regulatory sequences (promoters) that direct mRNA synthesis. In particular, examples of such transcriptional regulatory sequences suitable for use in transgenic plants include the cauliflower mosaic virus (CaMV), figwort mosaic virus (FMV), and strawberry bain banding virus (SVBV) promoters. , US provisional patent application No. 60 / 245,354. Prokaryotic cells, or eukaryotic cells, or other promoters known to regulate gene expression in their viruses, and promoters that can be used in some embodiments of the invention include the SV40 promoter, E.I. coli lac promoter or trp promoter, phage λPLPromoters can be mentioned. The expression vector optionally contains a ribosome binding site for translation initiation and a transcription terminator. The vector also optionally contains a suitable sequence (eg, enhancer) for amplifying expression. In addition, the expression vectors of the invention provide one or more selectable marker genes (eg, dihydrofolate reductase for eukaryotic cell culture or) to provide phenotypic traits for selecting transformed host cells. Neomycin resistance, or tetracycline resistance or ampicillin resistance within E. coli) is included as required.

The vectors of the invention can be utilized to transform a suitable host in order for the host to express the proteins or polypeptides of the invention. Examples of suitable expression hosts are: bacterial cells (eg E. coli, B. subtilis, Streptomyces, and Salmonella typhimurium); fungal cells (eg Saccharomyces cerevisiae, Pichia pastoris and Neurospora crassa); insect cells (eg Drosophila). And Spodoptera frugiperda); mammalian cells (eg, CHO, COS, BHK, HEK 293 or Bowes melanoma; or plant cells or explants, etc., all of the cells or cell lines are fully functional GAT poly It is understood that it is not necessary to have the ability to produce peptides; for example, antigenic fragments of GAT polypeptides can be produced. The invention is not limited by the host cell used.

In bacterial systems, many expression vectors can be selected depending on the intended use of the GAT polypeptide. For example, if the majority of GAT polypeptides or fragments thereof are required for commercial production or antibody induction, a vector may be desired that directs high level expression of the readily purified fusion protein. Such vectors include multifunctional E. coli cloning vectors such as BLUESCRIPT (Stratagene) and expression vectors (here, the coding sequence of the GAT polypeptide is such that the coding sequence of the GAT polypeptide expresses the hybrid protein at the amino terminus of β-galactosidase. Met followed by 7-residue sequence, which can be linked to the vector in frame); pIN vector (Van Heeke and Schuster (1989) J Biol Chem 264: 5503-5509); pET vector (Novagen, Madison WI), etc. However, it is not limited to them.

Similarly, in yeast Saccharomyces cerevisiae, many vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase and PGH, can be used for the production of GAT polypeptides of the invention. For a review, see Ausubel et al. (Supra) and Grant et al. (1987; Methods in Enzymology 153: 516-544).

Various expression systems can be used in mammalian host cells, including virus-based systems. When adenovirus is used as an expression vector, the coding sequence (eg, of a GAT polypeptide) can be optionally ligated into an adenovirus transcription / translation complex consisting of a late promoter and a tripartite leader sequence. Insertion of the GAT polypeptide coding region into the non-essential E1 or E3 region of the viral genome yields a viable virus capable of expressing GAT in infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci USA 81: 3655). -3659). In addition, transcription enhancers (eg, Rous sarcoma virus (RSV) enhancers) can be used to increase expression in mammalian host cells.

Similarly, in plant cells, expression can be driven in the cytoplasm from transgenes integrated into plant chromosomes or from episomal or viral nucleic acids. In the case of a stably integrated transgene, for example, a viral (eg, CaMV) or plant-derived control sequence can be used to drive a constitutive or inducible expression of the GAT polypeptide of the invention. It is often desired to provide. Many plant-derived regulatory sequences have been described, including sequences that direct expression in a tissue-specific manner (eg, TobRB7, Patatin B33, GRP gene promoter, rbcS-3A promoter, etc.). Alternatively, high level expression can be achieved by transiently expressing an exogenous sequence of a plant viral vector (eg, TMV, BMV, etc.). Typically, transgenic plants that constitutively express the GAT polynucleotides of the invention are preferred, and control sequences are selected to ensure constitutive and stable expression of GAT polypeptides.

In some embodiments of the invention, GAT polynucleotide constructs suitable for plant cell transformation are prepared. For example, the desired GAT polynucleotide can be incorporated into a recombinant expression cassette that facilitates gene transfer into the plant and subsequent expression of the encoded polypeptide. The expression cassette was operably linked to a promoter sequence and other transcription and translation initiation control sequences that direct expression of this sequence in the intended tissue of the transformed plant (eg, whole plant, leaves, seeds). It typically comprises a GAT polynucleotide or a functional fragment thereof.

For example, strong or weak constitutive plant promoters that direct the expression of GAT polypeptides in all plant tissues can be used. Such promoters are active under most environmental conditions and under developmental or cell-differentiated conditions. Examples of constitutive promoters include the 1'-or 2'-promoter derived from the T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions derived from various plant genes known to those of skill in the art. In situations where overexpression of GAT polynucleotides is harmful to the plant or otherwise undesired, one of ordinary skill in the art will appreciate that weak constitutive promoters can be used for low levels of expression. to understand. If high levels of expression are not harmful to the plant, a strong promoter (eg, t-RNA or other pol III promoter, or a strong pol II promoter (eg, cauliflower mosaic virus promoter)) can be used.

Alternatively, the plant promoter can be under environmental control. Such promoters are referred to herein as "inducible" promoters. Examples of environmental conditions that can result in transcription by an inducible promoter include pathogen attack, anaerobic conditions or the presence of light.

The promoters used in the present invention can be "tissue-specific" and can themselves be under developmental control in which polynucleotides are expressed only in specific tissues (eg, leaves and seeds). In embodiments where one or more nucleic acid sequences endogenous to the plant system are incorporated into the construct, an endogenous promoter (or variant thereof) derived from these genes is used to direct gene expression in the transfected plant. Can be used. Tissue-specific promoters can also be used to direct the expression of heterologous polynucleotides.

In general, the particular promoter used for an expression cassette in a plant depends on the intended use. Many promoters that direct transcription in plant cells are all suitable. Promoters can be either constitutive or inducible. In addition to the promoters described above, microbial promoters engineered in plants include the Octopin synthase promoter, the Noparin synthase promoter and other promoters derived from the native Ti plasmid (Herrara-Estrella et al. (1983) Nature 303). See: 209-213). Cauliflower mosaic virus 35S and 19S RNA promoters as virus promoters (Odell et al. (1985) Nature 313: 810-812). Other plant promoters include the ribulose-1,3-bisphosphate carboxylase small subunit promoter and the phaseolin promoter. Promoter sequences derived from the E8 gene and other genes can also be used. The isolation and sequence of the E8 promoter is described in detail in Deikman and Fischer (1988) EMBO J. 7: 3315-3327.

To identify candidate promoters, the 5'part of the genomic clone is analyzed for sequences characteristic of the promoter sequence. For example, the promoter sequence element includes the TATA box consensus sequence (TATAAT), which is usually 20 to 30 bases upstream of the transcription initiation site. In plants, further upstream of the TATA box, at positions -80 to -100, there is a typical promoter element with a series of adenines surrounded by three G (or T) nucleotides, which is described by Messing et al. (1983). ) Genetic Engineering in Plants, Kosage et al. (Ed.), Pp. 221-227.

Sequences other than promoters and ligated polynucleotides can be used in the preparation of the polynucleotide constructs of the invention (eg, vectors). If normal polypeptide expression is desired, a polyadenylation region may be included at the 3'end of the GAT coding region. The polyadenylation region can be, for example, from various plant genes or from T-DNA.

The construct may also contain a marker gene that imparts a selectable phenotype in plant cells. For example, markers can encode killing agent resistance, especially antibiotic resistance (eg, canamycin resistance, G418 resistance, bleomycin resistance, hyglomycin resistance), or herbicide resistance (eg, chlorosluforon or phosphino). It can encode tricin (the active ingredient of the herbicides bialaphos and Basta).

The particular initiation signal can aid in efficient transcription of the sequence encoding the GAT polynucleotide of the invention. These signals may include, for example, the ATG start codon and adjacent sequences. If the GAT polynucleotide coding sequence, its start codon and upstream sequence are inserted into a suitable expression vector, no further translational control signals may be required. However, if the coding sequence (eg, the mature protein coding sequence), or only a portion thereof, is inserted, an exogenous transcriptional control signal containing the start codon must be provided. In addition, the start codon must be present in the correct reading frame to ensure transcription of the entire insert. The extrinsic transcription elements and start codons can be of various origins and can be both natural and synthetic. Expression efficiency can be enhanced by encapsulating the appropriate enhancer in the cell line used (Scharf D et al. (1994) Results Probl Cell Differ 20: 125-62; Bittner et al. (1987) Methods in Enzymol 153: 516-544).

(Secreted / localized sequence) For example, to target polypeptide expression in the desired cellular compartment, membrane or organelle of mammalian cells, or to direct the secretion of polypeptide into the pericytoplasmic cavity or cell culture medium. The polynucleotides of the invention can also be fused in-frame to nucleic acids encoding secretory / localized sequences. Such sequences are known to those of skill in the art, and include secretory leader peptides, organelle targeting sequences (eg, nuclear localization sequences, ER retention signals, mitochondrial translocation sequences, chloroplast translocation sequences). Membrane localization / anchor sequences (eg, termination transition sequences, GPI anchor sequences) and the like.

In a preferred embodiment, the polynucleotides of the invention are in-frame with an N-terminal chloroplast transport sequence (or chloroplast transport peptide sequence) derived from a gene encoding a polypeptide normally targeted to chloroplasts. Fused. Such sequences are typically abundant in serine and threonine, deleted in aspartic acid, glutamic acid, and tyrosine, and generally have a rich central domain in positively charged amino acids.

(Expression host) In a further embodiment, the invention relates to a host cell containing the above constructs. The host cell can be a eukaryotic cell (eg, a mammalian cell, a yeast cell, or a plant cell), or the host cell can be a prokaryote (eg, a bacterial cell). Introduction of the construct into host cells can be performed by calcium phosphate transfection, DEAE-dextran-mediated transfection, electroporation, or other common techniques (Davis, L., Divner, M. and Battey, I. (1986) Basic Methods in Molecular Biology).

Host cell lines are optionally selected for their ability to regulate expression of the inserted sequence or process proteins expressed in the desired manner. Such modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves the "pre" or "prepro" form of a protein can also be important for accurate insertion, folding and / or function. Different host cells such as E. coli, Bacillus sp., Yeast or mammalian cells such as CHO, HeLa, BHK, MDCK, 293, WI38 have, for example, specific cellular and characteristic mechanisms for post-translational activity. And can be selected to ensure the desired modification and processing of the introduced foreign protein.

A stable expression system can be used for long-term, high-yield production of recombinant proteins. For example, a plant cell, implant or tissue (eg, shoot, leaf disk) that stably expresses the polypeptide of the invention can be expressed using an expression vector containing a viral origin of replication or an endogenous expression element and a selectable marker gene. And transduce. Depending on the cell type (eg, 1 hour or longer for bacterial cells, 1-4 days for plant cells, 2-4 weeks for several plant implants, after vector induction and before switching cells to selective medium. ) Can be grown in concentrated medium. The purpose of a selectable marker is to confer resistance to selection, and its presence allows the proliferation and recovery of cells that successfully express the introduced sequence. For example, transgenic plants expressing the polypeptides of the invention can be directly selected for resistance to the herbicide glyphosate. Stable transformed graft-derived resistant embryos can be propagated, for example, using tissue culture techniques appropriate for their cell type.

Host cells transformed with the nucleic acid sequence encoding the polypeptide of the invention are optionally cultured under conditions suitable for expressing and recovering the encoded protein from the cell culture. The protein or fragment thereof produced by the recombinant cell can be secreted, membrane-bound, or contained intracellularly, depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, expression vectors containing the GAT polynucleotides of the invention can be designed using a signal sequence that directs the secretion of a mature polypeptide through the prokaryotic or eukaryotic membrane.

(Additional polypeptide sequence) The polynucleotides of the invention may also include, for example, a coding sequence fused in-frame to a marker sequence that facilitates purification of the encoded polypeptide. Domains that facilitate such purification include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, sequences that bind to glutathione (eg, GST), hemagglutinin (eg, GST). HA) tag (corresponding to epitope derived from influenza hemagglutinin protein; Wilson et al. (1984) Cell 37: 767), Martose-binding protein sequence, FLAG epitope (Immunex) utilized in FLAGS expression / affinity purification system Corp, Seattle, WA), etc., but not limited to these. Inclusion of protease-cleavable polypeptide linker sequences between the purification domain and the GAT homolog sequence is useful for facilitating purification. One expression vector intended for use in the compositions and methods described herein is the expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site. I will provide a. Histidine residues facilitate purification for IMIAC (Porath et al. (1992) Protein Expression and Purification Immobilized metal ion affinity chromatography as described in 3: 263-281), enterokinase cleavage sites provide a means for separating GAT homolog polypeptides from fusion proteins. The pGEX vector (Promega; Madison, WI) can also be used to express foreign polypeptides as a fusion protein with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption on ligand-agarose beads (eg, glutathione-agarose in the case of GST-fusion), followed by the presence of free ligand. Can be eluted below.

(Polypeptide production and recovery) After transduction of the appropriate host strain and proliferation of the host strain for the appropriate cell density, the selected promoter is induced by appropriate means (eg, temperature change or chemical induction) and the cells are cultured for an additional period of time. Cells were typically collected by centrifugation, destroyed by physical or chemical means, and the resulting crude extract was retained for further purification. Microbial cells used in protein expression can be destroyed by any convenient method, including freezing-thaw cycles, sonication, mechanical destruction, or the use of cell lysates, or other methods. The method is well known to those skilled in the art.

As described, numerous references are available for culturing and producing a large number of cells, including cells of bacterial, plant, animal (especially mammalian) and archaeal origin. For example, Sambrook, Ausubel and Berger (all above), and Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3rd Edition, Wiley-Liss, New York and references cited therein; Doyle. and Griffiths (1997) Mammalian Cell Culture: Essential Techniques, John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, 4th Edition, WH Freeman and Company; and Ricciardelli et al. (1989) In vitro Cell See Dev.Biol.25: 1016-1024. For plant cell culture and redifferentiation, Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley & Sons, Inc., New York, NY; Gamborg and Phillips (ed.) (1995) Plant Cell, Tissue and Organ Culture. Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York); Jones (eds.) (1984) Plant Gene Transfer and Expression Protocols, Humana Press, Totowa, New Jersey and Plant Molecular Biology (1993) RRD Royale, Bios Scientific Publishers, Oxford, UKISBN0 12 198370 6. Common cell culture media are disclosed in Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL. Further information on cell culture can be found in commercial literature such as Life Sience Research Cell Culture Catalog (1998) ("Sigma-LSRCCC") from Sigma-Aldrich, Inc (StLouis, MO), and eg. Also found in The Plant Culture Catalog and supplement (1997) ("Sigma-PCCS") from Sigma-Aldrich, Inc (StLouis, MO). Further details regarding plant cell transformation and transgenic plant products are found below.

The polypeptides of the invention can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, such as ammonium sulfate or ethanol precipitation, acidic extraction, anions or cations. Exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (eg, using one of the tagging systems described herein), hydroxylapatite chromatography, and lectin chromatography Can be mentioned. As desired, a protein refolding step can be used to complete the conformation of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used in the final purification step. In addition to the references mentioned above, various purification methods are well known in the art, Sandana (1997) Bioseparation of Preteins, Academic Press, Inc .; and Bollag et al. (1996) Protein Methods, 2nd Edition, Wiley- Liss, NY; Walker (1996) The Pretein Protocols Handbook, Humana Press, NJ, Harris and Angal (1990) Protein Purification Applications: A Practical Approach, IRL Press at Oxford, Oxford, England; Harris and Angal, Protein Purification Methods: A Practical Approach, IRL Press at Oxford, Oxford , England; Scopes (1993) Protein Purification: Principles and Practice, 3rd Edition, Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, 2nd Edition, Wiley-VCH, NY; And Walker (1998) Protein Includes methods disclosed on Protocols on CD-ROM, Humana Press, NJ.

In some cases, it is desirable to produce the GAT polypeptides of the invention on a large scale, suitable for industrial and / or commercial applications. In such cases, the bulk fermentation procedure is used. Briefly, GAT polynucleotides (eg, polynucleotides containing any one of SEQ ID NOs: 1-5 and 11-262, or other nucleic acids encoding GAT polypeptides of the invention) are cloned into an expression vector. Can be transformed into. For example, US Pat. No. 5,955,310; Widner et al., "METHODS FOR PRODUCING A POLYPEPTIDE IN A BACILLUS CELL," describes a tandem promoter-bearing vector and a stabilized sequence operably linked to a polypeptide coding sequence. After inserting the polypeptide of interest into the vector, the vector is bacterial (eg, Bacillus subtilis). PL1801IIE strain (amyE, apr, npr, spoIIE :: Tn917) is converted to a host. Introduction of expression vectors into Bacillus cells is performed, for example, by proto-transformation (see, eg, Chang and Cohen (1979) Molecular General Genetics 168: 111) or by using competent cells (eg, Young and Spizizin (eg, Young and Spizizin). 1961) Journal of Bacteriology 81: 823 or Dubunau and Davidoff-Abelson (1971) Journal of Molecular Biology 56: 209), or by electroporation (see, eg, Shigekawa and Dower (1988) Biotechniques 6:742), Alternatively, it is achieved by joining (see, eg, Koehler and Thorne (1987) Journal of Bacteriology 169: 5271), and by Ausubel, Sambrook and Berger (all above).

Transformed cells are cultured in a nutrient medium suitable for the production of the polypeptide using methods known in the art. For example, cells are placed in a laboratory or suitable medium by shake flask culture or small or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentation). And can be cultured in an industrial fermenter carried out under conditions where the polypeptide can be expressed and / or isolated. Culturing is carried out in a suitable nutrient medium containing carbon and nitrogen sources as well as inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions (eg, in the American Type Culture Collection catalog). The secreted polypeptide can be recovered directly from the medium.

The obtained polypeptide can be isolated by a method known in the art. For example, the polypeptide can be isolated from the nutrient medium by conventional procedures, including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. The isolated polypeptide can then be further purified by various methods known in the art, such as chromatography (eg, ion exchange, affinity, hydrophobicity, isoelectric focusing, etc.). And size exclusion), electrophoresis procedures (eg, isoelectric focusing for preparative work), differential dissolution (eg, ammonium sulfate precipitation), or extraction (eg, Bollag et al. (1996) Protein Methods, 2nd edition, Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook, Humana Press, NJ; Bollag et al. (1996) Protein Methods, 2nd Edition, Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook, Humana Press, NJ), but not limited to these.

Cell-free transcription / translation systems can also be used to produce polypeptides using the DNA or RNA of the invention. Several such systems are commercially available. A general guide to in vitro transcription and translation protocols can be found in Tymms (1995) In vitro Transcription and Translation Protocols: Methods in Molecular Biology, Vol. 37, Garland Publishing, NY.

(Substituates and formats for sequence recombination) The polynucleotides of the invention are their use in standard cloning methods, such as those described by Ausubel, Berger and Sambrook, i.e. those for producing additional GAT polynucleotides and polypeptides with the desired properties. In addition to use, it is optionally used as a substrate for a variety of production procedures (eg, mutation, recombination, recombination reactions). A variety of generation protocols are available and described in the art. These procedures can be used separately and / or in combination to produce one or more variants of the polynucleotidase or polynucleotide set, as well as the encoded protein variants. Individually and collectively, these procedures are useful, for example, for the technology or rapid evolution of polynucleotides, proteins, pathways, cells and / or organisms with new and / or improved features. Provides a robust and widely applicable production method for diversified polynucleotides and sets of polynucleotides (eg, including polynucleotide libraries). The sequence-altering process can result, for example, single-base conversion, multi-base conversion, and insertion or deletion of nucleic acid sequence regions.

For clarity, distinctions and classifications are made in the discussion below, but it is often recognized that the techniques are not mutually exclusive. In fact, various methods can be used alone or in combination, in parallel or in succession, to obtain a variety of sequence variants.

The result of any of the diversity-producing procedures described herein can be the production of one or more polynucleotides, which are proteins that have or impart the desired properties. The polynucleotide encoding can be selected or screened. Following diversification by one or more of the methods herein or those available to those of skill in the art, any polynucleotide produced will have the desired activity or properties (eg, variable Km of glyphosate, acetyl-CoA). Variable Km, the use of alternative cofactors with increased kcat (eg, propionyl-CoA), etc. can be selected, for example, in an automated or configurable form, detected by either an assay in the art. It may include identifying any activity to obtain. For example, GAT homologs with increased specific activity are detected by assaying the conversion of glyphosate to N-acetyl glyphosate (eg, by mass analysis). Alternatively, an improved ability to confer resistance to glyphosate can be obtained by culturing bacteria transformed with the nucleic acids of the invention on agar (including increased concentrations of glyphosate), or with glyphosate the nucleic acids of the invention. It can be assayed by spraying the incorporated transgenic plants. Various related properties (or even unrelated properties) can be evaluated continuously or in parallel at the discretion of the practitioner. Further details regarding recombination and selection for resistance of herbicides can be found, for example, in "DNA SHUFFLING TO PRODUCE HERBICIDE RESISTANT CROPS" (filed August 12, 1999) (USSN 09 / 373,333).

Details of the various diversity generation procedures include family shuffling and methods for generating modified nucleic acid sequences encoding multiple enzyme regions, found in the following publications and references cited therein: Soong. , N. et al. (2000) "Molecular breeding of viruses" Nat Genet 25 (4): 436-39; Stemmer et al. (1999) "Molecular breeding of viruses for targeting and other clinical properties" Tumor Targeting 4: 1-4; Ness Et al. (1999) "DNA Shuffling of subgenomic sequences of subtilisin" Nature Biotechnology 17: 893-896; Chang et al. (1999) "Evolution of a nucleic acid using DNA family shuffling" Nature Biotechnology 17: 793-797; Minshull and Stemmer (1999) "Protein evolution by molecular breeding" Current Opinion in Chemical Biology 3: 284-290; Christians et al. (1999) "Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling" Nature Biotechnology 17: 259-264; Crameri et al. (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution" Nature 391: 288-291; Crameri et al. (1997) "Molecular evolution of an arsenate detoxification pathway" by DNA shuffling "Nature Biotechnology 15: 436-438; Zhang et al. (1997)" Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening "Proc. Natl. Acad. Sci. USA 94: 4504-4509; Patten et al. (1997) "Applications of DNA Shuffling to Pharmaceuticals and Vaccines" Current Opinion in Biotechnology 8: 724-733; Crameri et al. (1996) "Construction and evolution of antibody-phage libraries by DNA shuffling" Nature Medicine 2: 100-103; Crameri Et al. (1996) "Improved green fluorescent protein by molecular evolution using DNA shuffling "Nature Biotechnology 14: 315-319; Gates et al. (1996)" Affinity selective isolation of ligands from peptide libraries through display on a lac repressor "headpiece dimer" Journal of Molecular Biology 255: 373- 386; Stemmer (1996) "Sexual PCR and Assembly PCR": The Encyclopedia of Molecular Biology.VCH Publishers, New York.pp.447-457; Crameri and Stemmer (1995) "Combinatorial multiple cassette mutagenesis creates" "All the permutations of mutant and wildtype cassettes" BioTechniques 18: 194-195; Stemmer et al. (1995) "Single-step assembly of a gene and entire plasmid form large numbers of oligodeoxy-ribonucleotides" Gene, 164: 49-53; Stemmer ( 1995) "The Evolution of Molecular Computation" Science 270: 1510; Stemmer (1995) "Searching Sequence Space" Bio / Technology 13: 549-553; Stemmer (1994) "Rapid evolution of a protein in vitro by DNA shuffling" Nature 370 : 389-391; and Stemmer (1994) "DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc. Natl Acad.Sci.USA 91: 10747-10751.

Mutagenesis methods that generate diversity include, for example: site-specific mutagenesis (Ling et al. (1997) "Approaches to DNA mutagenesis: an overview" Anal. Biochem. 254 (2): 157-178. Dale et al. (1996) "Oligonucleotide-directed random mutagenesis using the phosphorothioate method" Methods Mol.Biol. 57: 369-374; Smith (1985) "In vitro mutagenesis" Ann. Rev. Genet. 19: 423-462; Botstein & Shortle (1985) "Strategies and applications of in vitro mutagenesis" Science 229: 1193-1201; Carter (1986) "Site-directed mutagenesis" Biochem. J. 237: 1-7; and Kunkel (1987) "The efficiency of oligonucleotide directed mutagenesis "Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, DMJ., Springer Verlag, Berlin)); Mutagenesis with uracil containing templates (Kunkel (1985)" Rapid and efficient site- "Specific mutagenesis without phenotypic selection" Proc. Natl.Acad.Sci.USA 82: 488-492; Kunkel et al. (1987) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Methods in Enzymol. 154,367-382; and Bass et al. 1988) "Mutant Trp repressors with new DNA-binding specificities "Science 242: 240-245); Oligonucleotide specific mutagenesis (Methods in Enzymol. 100: 468-500 (1983); Methods is Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)" Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment "Nucleic Acids Res. 10: 6487-6500; Zoller & Smith (1983)" Oligonucleotide-directed mutagenesis of DNA fragments " cloned into M13 vectors "Methods in Enzymol. 100: 468-500; and Zoller & Smith (1987) "Oligonucleotide-directed mutagenesis: a simple methods using two oligonucleotide primers and a single-stranded DNA templete" Methods in Enzymol. 154: 329-350); Mutation induction (Taylor et al. (1985) "The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA" Nucl. Acids Res. 13: 8749-8764; Taylor et al. (1985) "The rapid generation of oligonucleotide-directed mutations" at high frequency using phosphorothioate-modified DNA "Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye & Eckstein (1986)" Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis "Nucl.Acids Res. 14: 9679-9698; Sayers et al. (1988) "YT Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis" Nucl. Acids Res. 16: 791-802; and Sayers et al. (1988) "Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidiurm bromide "Nucl. Acids Res. 16: 803-814); Kramer et al. (1984)" The gapped duplex DNA approach to oligonucleotide-directed mutations construction Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol Oligonucleotide-directed construction of mutations via gapped duplex DNA 154.350-367; Kramer et al. the gapped duplex DNA approach to oligonucleotide-directed construction of mutations "Nucl. Acids Res. 16: 7207; and Fritz et al. (1988)" Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without resonator reactions in vitro "Nucl. Acids Res. 16.6987-6999) ..

Further suitable methods include: point mismatch repair (Kramer et al. (1984) "Point Mismatch Repair" Cell 38: 879-887), mutagenesis using repair defective host strains (Carter et al. (1985)). "Improved oligonucleotide site-directed mutagenesis using M13 vectors" Nucl. Acids Res. 13: 4431-4443; and Carter (1987) "Improved oligonucleotide-directed mutagenesis using M13 vectors" Methods in Enzymol. 154: 382-403), deletion Mutation Induction (Eghtedarzadeh & Henikoff (1986) "Use of oligonucleotides to generate large deletions" Nucl. Acids Res.14: 5115), Restriction-Selection and Restriction-Purification (Wells et al. (1986) "Importance of hydrogen-bond formation in stabilizing the transition state of subunit" Phil.Trans.R.Soc.Lond.A 317: 415- 423), Mutation induction by total gene synthesis (Nambiar et al. (1984) "Total synthesis and cloning of a gene coding for the ribonuclease S protein" Science 223: 1299-1301; Sakamar and Khorana (1988) "Total synthesis and expression of a" gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin) "Nucl. Acids Res. 14: 6361-6372; Wells et al. (1985)" Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites "Gene 34: 315-323; and Grundstrom et al. (1985) "Oligonucleotide-directed mutagenesis by microscale'shot-gun' gene synthesis" Nucl. Acids Res. 13: 3305-3316), Double-strand break repair (Mandecki (1986); Arnold (1993) "Protein engineering for unusual environments" Current Opinion in Biotechnology 4: 450-455. "Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis "Proc. Natl. Acad. Sci. USA, 83: 7177-7181). Further details on many of the above methods can be found in Methods in Enzymology, Vol. 154, which also describes useful controls for troubleshooting problems with various mutagenesis methods.

Further details on various methods of generating diversity can be found in the following US patents, PCT publications, and EP publications: US Pat. No. 5,605,793 to Stemmer (February 25, 1997), "Methods for In vitro Recombination." "Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" US Pat. No. 5,811,238 to Stemmer et al. (November 3, 1998), "Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" DNA Mutagenesis by Random Fragmentation and Reassembly "; US Pat. No. 5,834,252 for Stemmer (November 10, 1998)" End-Complementary Polymerase " Reaction; US Pat. No. 5,837,458 for Minshull (17 November 1998), Methods and Compositions for Cellular and Metabolic Engineering; WO 95/22625, Stemmer and Crameri, Mutagenesis by Random Fragmentation and Reassembly; Stemmer and WO 96/33207 by Lipschutz, "End Complementary Polymerase Chain Reaction;" WO 97/20078 by Stemmer and Crameri, "Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" WO 97/35966 by Minshull and Stemmer, "Methods" "And Compositions for Cellular and Metabolic Engineering;" WO 99/41402 by Punnonen et al., "Targeting of Genetic Vaccine Vectors;" WO 99/41383 by Punnonen et al., "Antigen Library Immunization;" WO 99/41369 by Punnonen et al., "Genetic Vaccine" Vector Engineering; "WO 99/41368 by Punnonen et al.," Optimization of Immunomodulatory Properties of Genetic Vaccines; "EP 752008 by Stemmer and Crameri," DNA Mutagenesis by Random Fragmentation and Reassembly; "EP 0932670 by Stemmer," Evolving Cellular DNA Uptake by Recursive Sequence Recombination; "WO 99/23 107 by Stemmer et al.," Modification of Virus Tropism and Host Range by Viral Genome Shuffling; "WO 99/21979 by Apt et al.," Human Papillomavirus Vectors; "WO 98/31837 by del Cardayre et al. , "Evolution of Whole Cells and Organisms by Recursive Sequence Recombination;" WO 98/27230 by Patten and Stemmer, "Methods and Compositions for Polypeptide Engineering;" WO 98/13487 by Stemmer et al., "Methods for Optimization of Gene Therapy by Recursive" Sequence Shuffling and Selection "WO 00/00632," Methods for Generating Highly Diverse Libraries, "WO 00/09679," Methods for Obtaining in Vitro Recombined Polynucleotide Sequence Banks and Resulting Sequences, "Arnold et al. Using Random or Defined Primers, "WO 99/29902 by Arnold et al.," Method for Creating Polynucleotide and Polypeptide Sequences, "WO 98/41653 by Vind," An in Vitro Method for Construction of a DNA Library, "WO 98/41622 by Borchert et al.," Method for Constructing a Library Using DNA Shuffling, "and WO 98/42727 by Pati and Zarling," Sequence Alterations using Homologous Recombination "WO 00/18906 by Patten et al.," Shuffling of Codon- "Altered Genes;" WO 00/04190 by del Cardayre et al., "Evolution of Whole Cells and Organisms by Recursive Recombination;" WO 00/42 561, by Crameri et al. Methods of Populating Data "Structure for Use in Evolutionary Simulations;" WO 00/42560 by Serifonov et al., "Methods for Making Character Strings, Polynucleotides & Polypeptides Having Desired Characteristics;" WO 01/23401 by Welch et al., "Use of Codon-Varied Oligonucleotide Synthesis for Synthetic Shuffling;" And PCT / US 01/06775, "Single-Stranded Nucleic Acid Template-Mediated Recombination and Nucleic Acid Fragment Isolation" by Affholter.

Certain US applications provide further details on various methods of generating diversity, including: "SHUFFLING OF CODON ALTERED GENES" (Patten et al., September 28, 1999) (USSN 09 / 407,800); "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION" del (Cardayre et al., Filed July 15, 1998) (USSN09 / 166,188), and July 15, 1999, (USSN09 / 354,922); "OLIGONUCLEOTIDE" "MEDIATED NUCLEIC ACID RECOMBINATION" (Crameri et al., Filed September 28, 1999) (USSN09 / 408,392); "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" (Crameri et al., filed January 18, 2000) (PCT / US00 / 01203); "USE OF CODON-BASED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING "(Welch et al., Filed September 28, 1999) (USSN09 / 408,393);" METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS "(Selifonov et al., Filed January 18, 2000) (PCT / US00 / 01202);" METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS (USSN / 09 / 618,579) and "METHODS OF POPULATING DATA STRUCTURES FOR USE IN EVOLUTIONARY SIMULATIONS" (Selifonov and Stemmer et al., Filed January 18, 2000) (PCT / US00 / 01138) "SINGLE-STRANDED NUCLEIC ACID" TEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION Affholter (USSN 60 / 186,482, filed February 2, 2000).

In summary, general classes of several different sequence modification methods, such as mutation, recombination, etc., are applicable to the present invention and are described, for example, in the references above. That is, modifications of the component nucleic acid sequence to the produced modified gene fusion construct can be performed either before the cojoining of the sequence or after the binding step by a number of protocols described. In the context of the present invention, some of the following exemplary different types of preferred forms for making diversity include, for example, specific recombination based on the form of making diversity.

Nucleic acids can be recombined in vitro by any of the various techniques discussed in the references above, including, for example, ligation and / or PCR reconstruction of the nucleic acid following DNAse digestion of the recombined nucleic acid. For example, sequence similarity-based recombination between DNA molecules with different but related DNA sequences in vitro, followed by random (or pseudo-random, or even non-random) fragmentation of DNA molecules, followed by polymerase chain reaction. PCR mutagenesis for sex can be used by fixation of crossings by elongation in the reaction. This process and variations of many processes are described in some of the above references (eg, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751).

Similarly, nucleic acids can be recombinated in vitro, for example by causing recombination between intracellular nucleic acids. Many such forms of in vitro recombination are described in the references described above. Such a form provides direct recombination between the nucleic acids of interest, if desired, or, like any other form, between vectors, viruses, plasmids, etc. containing the nucleic acids of interest. Provide recombination. Details regarding such procedures can be found in the references above.

The entire genome of a cell or other organism is recombined, including spiking of a recombinant mixture of genomes having the desired library components (eg, genes corresponding to the pathways of the invention) as needed. Genome-wide recombination methods can also be used. These methods have many uses, including those for which the identity of the target gene is unknown. For more information on such methods, see, for example, "Evolution of Whole Cells and Organisms by Recursive Sequence Recombination" in WO 98/31837 by del Cardayre et al .; and, for example, PCT / US99 / 15972 by del Cardayre et al. Evolution of Whole Cells and Organisms by Recursive Sequence Found in "Recombination". Thus, any of these processes and techniques for recombination, recombination, and genome-wide recombination, alone or in combination, have modified nucleic acid sequences and / or modified genes of the invention. It can be used to produce fusion constructs.

A synthetic recombination method in which an oligonucleotide corresponding to a target of interest is synthesized and reconstituted in a PCR or ligation reaction containing an oligonucleotide corresponding to two or more parent nucleic acids, thereby producing a new recombinant nucleic acid. Can also be used. Oligonucleotides can be made by standard nucleotide addition methods or, for example, by a trinucleotide synthesis approach. Details on such an approach can be found in the references above, eg WO 00/42561 by Crameri et al., "Oligonucleotide (Olgonucleotide) Mediated Nucleic Acid Recombination"; WO 01/23401 by Welch et al., "Use of Codon-Varied". Oligonucleotide Synthesis for Synthetic Shuffling ; WO 00/42560 by Selifonov et al., Methods for Making Character Includes "Strings, Polynucleotides and Polypeptides Having Desired Characteristics"; and WO 00/42559 "Methods of Populating Data Structures for Use in Evolutionary Simulations" by Serifonov and Stemmer.

A method of recombination in in silico, in which a genetic algorithm is used in a computer to recombine a string of sequences corresponding to homologous (or even non-homologous) nucleic acids, can be achieved. The resulting string of the recombinant sequence is converted to nucleic acid, if necessary, for example, in combination with oligonucleotide synthesis / gene reconstruction techniques, by synthesizing the nucleic acid corresponding to the recombinant sequence. This approach can produce random, partially random or designed variants. Many details about recombination in Insilico include the production of the corresponding nucleic acid (and / or protein), and the combination of the designed nucleic acid and / or protein (eg, based on cross-position selection), as well as the design. WO 00/42560 by Seliponov et al., "Methods for Making Character Strings, Polynucleotides and Polypeptides Havings," including the use of genetic algorithms, genetic operators, etc. in computer systems combined with pseudo-random or random recombination methods. Described in "Desire Characteristics" and WO 00/42559 "Methods of Populating Data Structures for Use in Evolutionary Simulations" by Serifonov and Stemmer. Extensive details regarding recombination methods in silico are found in these applications. This methodology is generally associated with various metabolic pathways in insilico, such as carotinoid biosynthetic pathways, ectoine biosynthetic pathways, polyhydroxyalkanoate biosynthetic pathways, aromatic polyketide biosynthetic pathways, etc. It is applicable to the present invention in providing for the recombination of nucleic acid sequences and / or gene fusion constructs encoding proteins and / or the production of corresponding nucleic acids or proteins.

For example, by hybridization of a wide variety of nucleic acids or nucleic acid fragments to a single-stranded template, polymerization and / or ligation to regenerate the full-length sequence, and optionally by degradation of the template and recovery of the resulting modified nucleic acid. Many methods of utilizing natural diversity can be used as well. In one method utilizing a single-stranded template, a population of fragments from the genomic library is annealed using a portion of ssDNA or RNA corresponding to the contralateral strand, or often near full length. The assembly of complex chimeric genes from this population then removes the nucleobase at the ends of the non-hybridizing fragment, polymerization to fill the gap between such fragment and the resulting single chain ligation. Mediated by. The parental polynucleotide strand is digested (eg, if it contains RNA or uracil), magnetically separated under denatured conditions (if labeled in a manner that leads to such separation), and other usable separations / purifications. Can be removed by method. Alternatively, the parent strand is, if necessary, purified with the chimeric strand and removed during subsequent screening and processing steps. Further details on this approach can be found, for example, in PCT / US01 / 06775, "Single-Stranded Nucleic Acid Template-Mediated Recombination and Nucleic Acid Fragment Isolation" by Affholter.

In another approach, a single-stranded molecule is converted to double-stranded DNA (dsDNA), which is bound to a solid support by ligand-mediated binding. After separation of unbound DNA, the selected DNA molecule is released from the support and introduced into a host cell suitable for producing a library-rich sequence that hybridizes to the probe. .. The library produced in this method uses any of the procedures described herein to provide the desired substrate for further diversification.

Any of the above-mentioned common forms of recombination requires a repetitive mode (eg, one or more cycles of mutation / recombination or other diversity production method,) to produce a more diverse set of recombinant nucleic acids. Depending on, followed by one or more selection methods).

Mutation induction utilizing polynucleotide chain termination methods has also been proposed (eg, US Patent Application No. 5,965,408 by Short, "Method of DNA reassembly by interrupting". "Synthesis", and the references above), and can be applied to the present invention. In this approach, double-stranded DNA corresponding to one or more genes that share a region of sequence similarity is bound and denatured in the presence or absence of primers specific for that gene. Single-stranded polynucleotides are then such as polymerases and chain arrest reagents (eg, UV irradiation, γ-ray irradiation or X-ray irradiation; ethidium bromide or other intercalators; single-chain binding proteins, transcriptional activators, or histones. DNA binding proteins; polycyclic aromatic hydrocarbons; trivalent chromium or trivalent chromium salts; or simplified polymerization mediated by rapid thermal circulation; etc.) Annexed and incubated in the presence of results. Partially results in the production of double-stranded molecules. Partial duplex molecules, for example containing partially extended strands, then share varying degrees of sequence similarity and are diversified polynucleotides for the first population of DNA molecules. Is modified and reannealed in subsequent iterations of replication or partial replication resulting in. If desired, the product, or partial pool of product, can be amplified in one or more steps in this process. Polynucleotides produced by the chain termination method, as described above, are suitable substrates for any other described recombinant form.

Diversity is also described in Ostermeier et al. (1999) "A combinatorial approach to hybrid enzymes independent of DNA homology" Nature Biotech 17: 1205, "incremental truncation for the creation of hybrid." It can be produced in nucleic acids or populations of nucleic acids using a recombinant procedure called "enzymes" ("ITCHY"). This approach can optionally be used to produce an initial library of variants that can serve as a substrate for one or more in vitro or in vivo recombination methods. Ostermeier et al. (1999) "Combinatorial Protein Engineering by Incremental" "Truncation", Proc.Natl.Acad.Sci.USA, 96: 3562 ~ 67; Ostermeier et al. (1999), "Incremental Truncation as a Strategy in the Engineering of Novel Biocatalysts", Biological and Medicinal Chemistry, 7: 2139 ~ 44 See also.

Methods of mutation that result in modification of individual nucleotides or groups of contiguous or non-contiguous nucleotides are preferred to be used to introduce nucleotide diversity into the nucleic acid sequences and / or gene fusion constructs of the invention. Can be done. Many mutagenesis methods are found in the references described above; further details regarding mutagenesis methods can be found below, which can also be applied to the present invention.

For example, error-prone PCR can be used to produce nucleic acid variants. Using this technique, PCR is performed under conditions that reduce the accuracy of copying DNA polymerase, resulting in a high rate of point mutations along the overall length of the PCR product. Examples of such techniques are found in the references above and, for example, in Leung et al. (1989) Technique 1: 11-15 and Caldwell et al. (1992) PCR Methods Applic. 2: 28-33. Similarly, assembly PCR can be used in steps related to the assembly of PCR products from a mixture of small DNA fragments. Many different PCR reactions can occur simultaneously in the same reaction mixture, where the product of one reaction primes the product of another reaction.

Oligonucleotide-directed mutagenesis can be used to induce site-specific mutagenesis in a nucleic acid sequence of interest. Examples of such techniques are found in the above references and, for example, in Reidhaar-Olson et al. (1988) Science, 241: 53-57. Similarly, cassette mutagenesis can be used in the step of substituting small regions of a double-stranded DNA molecule with a cassette of synthetic oligonucleotides that differ from the native sequence. Oligonucleotides can include, for example, fully and / or partially randomized native sequences.

Recursive ensemble mutagenesis is the process by which algorithms for protein mutagenesis are used to produce a diverse population of phenotypically related variants, the members of which differ in amino acid sequence. This method uses a feedback mechanism to monitor the continuous repetition of combinatorial cassette mutagenesis. An example of this approach can be found in Arkin & Youvan (1992) Proc.Natl.Acad.Sci.USA 89: 7811-7815.

Exponential ensemble mutagenesis can be used to create combinatorial libraries with a high proportion of unique and functional variants. A small group of residues in the sequence of interest is randomly randomized in parallel for each modified position to identify the amino acid that results in the functional protein. Examples of such procedures are found in Delegrave & Youvan (1993) Biotechnology Research 11: 1548 ~ 1552.

In vivo mutagenesis is used, for example, in strains of E. coli that carry mutations in one or more DNA repair pathways to produce random mutations in any cloned DNA of interest by increasing the DNA. obtain. These "mutator" strains have a higher rate of random mutation than the rate of wild-type parents. Increasing DNA in one of these strains ultimately produces random mutations in the DNA. Such procedures are described in the references described above.

Other procedures for introducing diversity into the genome (eg, bacterial genome, fungal genome, animal genome or plant genome) can be used in conjunction with the methods described above and / or referenced methods. For example, in addition to the above methods, techniques for producing nucleic acid multimers suitable for transformation into various species have been proposed (see, eg, Schellenberger, U.S. Patent Application No. 5,756,316 and the above references. thing). Appropriate host transformations in such multimers are different from each other (eg, ranging from natural diversity or site-specific mutagenesis, error-prone PCR, mutagenic strains). It is constructed from genes (eg, by in vivo recombination steps as described above) that provide a source of nucleic acid diversity for DNA diversification (by passage, etc.).

Alternatively, the multiplicity of monomeric polynucleotides that share a region of partial sequence similarity can be transformed into the host species and recombined in vivo by the host cell. Subsequent repetition of cell differentiation can be used to produce the library, the members of the library comprising a pool of single, homologous populations, or monomeric polynucleotides. Alternatively, the monomeric nucleic acid can be recovered by standard techniques (eg, PCR and / or cloning) and in any form of recombination, including the forms of recombination described above. Can be recombined.

Methods for making a wide variety of expression libraries have been described (in addition to the references described above, for example, Peterson et al. (1998) US Patent Application No. 5,783,431 "METHODS FOR GENERATING AND SCREENING NOVEL METABOLIC PATHWAYS", And Thompson et al. (1998) US Patent Application No. 5,824,485 METHODS FOR GENERATING AND SCREENING NOVEL METABOLIC PATHWAYS), and their use in identifying the activity of the protein of interest has been proposed (references above). In addition to the literature, Short (1999) US Patent Application No. 5,958,672 "PROTEIN ACTIVITY SCREENING OF CLONES HAVING DNA FROM UNCULTIVATED" See MICROORGANI SMS). Multi-expression libraries generally include libraries containing cDNA or genomic sequences from multiple species or strains operably linked to appropriate regulatory sequences within an expression cassette. The cDNA and / or genomic sequences are randomly linked, if desired, to further enhance diversity. The vector can be a suitable shuttle vector for transformation and expression in more than one species of host organism (eg, bacterial species, eukaryotic cells). In some cases, the library is biased by preselecting a sequence that encodes the protein of interest or hybridizes to the nucleic acid of interest. Any such library may be provided as a substrate for any of the methods described herein.

The procedures described above have mostly been directed towards increasing the diversity of nucleic acids and / or the diversity of encoded proteins. However, in many cases, not all diversity is useful (eg, functionally) and simply increases the background of variants that must be screened or selected to identify the few preferred variants. It only contributes. In some applications, libraries (eg, amplified libraries, genomic libraries, cDNA libraries, standardized libraries, etc.) or diversified (eg, by recombination-based mutagenesis procedures). It is desirable to preselect or pre-screen the other previous nucleic acid substrates, or otherwise bias the substrate towards the nucleic acid encoding the functional product. For example, in the case of antibody design, prior to manipulation by any of the described methods, a step of utilizing the recombination phenomenon in vivo to produce diversity towards an antibody having a functional antigen binding site. It is possible to bias. For example, recombinant CDRs from B cell cDNA libraries can be amplified and constructed within framework regions prior to diversification according to any of the methods described herein (eg, Jirholt et al. (1998)). "Exploiting sequence space: shuffling in vivo complementarity determining regions into a master framework" Gene 215: 471).

The library can be biased towards the nucleic acid encoding the protein with the desired enzymatic activity. For example, after identifying a clone from a library that exhibits a particular activity, this clone can be mutated using any known method for introducing DNA alterations. The library containing the mutated homologue is then screened for the desired activity, which may be the same as or different from the first particular activity. An example of such a procedure is Short (1999) US Patent Application No. 5,939,250, "PRODUCTION OF ENZYMES HAVING DESIRED ACTIVITIES BY. Proposed during "MUTAGENESIS". The desired activity can be identified by any method known in the art. For example, WO99 / 10539 is screened by the gene library mixing extracts from the gene library with components obtained from metabolically abundant cells and identifying combinations that exhibit the desired activity. I'm proposing to get it. A clone with the desired activity inserts a bioactive substrate into a sample of the library and uses a fluorescence analyzer (eg, a flow cytometry device, CCD, fluorescence meter, or spectrophotometer). It has also been proposed that it can be identified by detecting the bioactive fluorescence corresponding to the product of the desired activity (eg WO98 / 58085).

The library can also be biased towards nucleic acids with specific properties (eg, hybridization to selected hybrid probes). For example, application WO99 / 10539 states that the desired activity (eg, enzymatic activity (eg: lipase, esterase, protease, glycosidase, glycosyltransferase, phosphatase, kinase, oxygenase, peroxidase, hydrolase, hydratase, nitrilase, transamidase, amidase, or acylase). It is proposed that the polynucleotide encoding)) can be identified from the genomic DNA sequence by the following method. Single-stranded DNA molecules from a population of genomic DNA hybridize to a ligand-binding probe. This genomic DNA can be derived from cultured or uncultured microorganisms, or samples in the environment. Alternatively, the genomic DNA can be derived from a multicellular organism, or a tissue derived from a multicellular organism. The second strand synthesis is performed directly from the hybridization probe used for capture, either released first from the capture medium or not, or by a wide variety of other methods known in the art. obtain. Alternatively, a population of isolated single-stranded genomic DNA can be fragmented without further cloning and, as described above, directly using a single-stranded template (eg, recombination). Approach based on) Can be used.

A "non-stochastic" method of producing nucleic acids and polypeptides is claimed in Short "Non-Stochastic Generation of Genetic Vaccines and Enzymes" WO 00/46344. These methods, including non-stochastic polynucleotide restructuring and site-saturation mutagenesis, apply similarly to the present invention. Random or semi-random mutagenesis using doped or degenerate oligonucleotides also includes, for example, Arkin and Youvan (1992) "Optimizing nucleotides to encode specific subsets of amino acids for semi-random mutagenesis" Biotechnology 10: 297 ~ 300; Reidhaar-Olson et al. (1991) "Random mutagenesis of protein sequences using oligonucleotides" cassettes "Methods Enzymol. 208: 564 ~ 86; Lim and Sauer (1991)" The role of internal packing interactions in determining the structure and stability of a protein "J. Mol. Biol. 219: 359 ~ 76; Breyer and Sauer (1991) 1989) "Mutational analysis of the fine specificity of binding of monoclonal antibody 51F to lambda repressor" J. Biol. Chem. 264: 13355-60; and "Walk-Through Mutagenesis" (Crea, R; US Patent Application No. 5,830,650 and It is described in US Patent Application No. 5,798,208 and European Patent Application No. 0527809 B1).

Any of the techniques described above, suitable for enriching the library prior to diversification, is also used to screen for products, or libraries of products, produced by methods that produce diversity. It is easily understood that it can be used. Any of the methods described above can be performed recursively or in combination to alter a nucleic acid (eg, a GAT-encoding polynucleotide).

Kits for mutagenesis methods, library construction methods and other diversity production methods are also commercially available. For example, the kit can be, for example, Stratagene (eg, QuickChange).TM Site-specific mutagenesis kit; and ChameleonTM Double-stranded site-specific mutagenesis kit), Bio / Can Scientific, Bio-Rad (eg, using the Kunkel method described above), Boehringer Mannheim Corp., Clonetech Laboratories, DNA Technologies, Epicentre Technologies (eg, 5). Prime 3 Prime Kit); Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), New England Biolabs, Pharmacia Biotech, Promega Corp., Quantum Biotechnologies, Amersham International plc (eg, using the Eckstein method above), and Anglian. It is available from Biotechnology Ltd (eg, using the Carter / Winter method described above).

The above references provide many forms of mutation, including: recombination, recombination, recombination, recombination, and combination or recombination with other forms of mutagenesis. , As well as many improvements in these formats. Regardless of the variety-producing form used, the nucleic acids of the invention are recombinant (with respect to, or with related sequences (or even unrelated sequences)), the gene fusion constructs of the invention and modified genes. Various sets of recombinant nucleic acids for use in fusion constructs can be produced, including, for example, sets of homologous nucleic acids and corresponding polypeptides.

Many of the above-mentioned methodologies for producing modified polynucleotides produce a large number of diverse variants of the parental sequence. In some preferred embodiments of the invention, a modification technique (eg, some form of shuffling) is used to create a library of variants, which is then used for some desired function. Screened for modified polynucleotides or pools of modified polynucleotides encoding specific properties (eg, improved GAT activity). An exemplary enzyme activity that can be screened is the catalytic rate (k).catAnd KMIncludes (conventionally characterized with respect to rate constants such as), substrate specificity, and susceptibility to activation or inhibition by a substrate, product or other molecule (eg, inhibitor or activator).

One example of selection for the desired enzyme activity is to grow the host cell under conditions that inhibit the growth and / or survival of cells that do not adequately express the desired enzyme activity (eg, GAT activity). Accompanied by. Using such a selection step, all modified polynucleotides other than the polynucleotide encoding the desired enzymatic activity can be excluded from consideration. For example, in some embodiments of the invention, the host cell is under conditions that inhibit cell proliferation or survival in the absence of sufficient levels of GAT (eg, lacking and not expressing GAT polynucleotides). Concentrations of glyphosate that are lethal or impede to the growth of wild-type plants of the variety) are retained. Under these conditions, only host cells with modified nucleic acids encoding enzymatic activity or activity that can catalyze the production of sufficient levels of product will survive and proliferate. Some embodiments of the invention use multiple repetitive screenings by increasing the concentration of glyphosate or glyphosate analog.

In some embodiments of the invention, mass spectrometry is used to detect acetylation of glyphosate or glyphosate analogs or metabolites. The use of mass spectrometry will be described in more detail in the examples below.

For convenience and high throughput, it is often desired to screen / select for the desired modified nucleic acid in a microorganism (eg, a bacterium such as E. coli). On the other hand, screening on plant cells or plants may be preferred in some cases. Here, the ultimate goal is to produce a modified nucleic acid for expression in a plant system.

In some preferred embodiments of the invention, the amount treated is increased by screening a pool of host cells expressing different modified nucleic acids, either alone or as part of a gene fusion construct. Any pool showing significant activity can be inversely superimposed to identify a single clone expressing the desired activity.

Those skilled in the art will recognize that the relevant assay, screening or selection method will vary depending on the desired host organism and the like. It is usually advantageous to use an assay that can be performed in a high throughput format.

In high-throughput assays, it is possible to screen thousands of different variants per day. For example, a separate assay can be performed using each well of the microtiter plate, or a single variant can be tested every 5-10 wells if the effect of concentration or incubation time is observed. ..

In addition to the fluid approach, as described above, it is possible to simply grow cells on a plate medium and select for the desired enzymatic or metabolic function. This approach provides a simple high-throughput screening method.

Several well-known automated system systems have also been developed for phase chemistry useful in assay systems. These systems are many automated equipment systems (Zymate II) that use automated synthesizers developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and robotic arms that mimic manual synthesis operations performed by scientists. Includes automated workstations such as Zymark Corporation, Hopkinton, MA .; Orca, Hewlett-Packard, Palo Alto, CA). Any of the above devices are suitable for application to the present invention. The features and implementation of improvements to these devices, if any, will be understood by those skilled in the art so that they can be operated as described herein with references to the integrated system. Will be done.

High-throughput screening systems are commercially available (eg, Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc. See). These systems typically automate the entire procedure, including: pipetting all samples and reagents, dispensing liquids, and incubating at designated times. The process, and the process of final loading of the microplate in a detector suitable for the assay. These structurable systems offer high throughput and rapid operation as well as a high degree of freedom and custom production.

Manufacturers of such systems provide detailed protocols for various high-throughput devices. Thus, for example, Zymark Corp. provides technical notices describing screening systems for detection of gene transcript regulation, ligand binding, and the like. A microfluidic approach to reagent manipulation has been developed, for example, by Caliper Technologies (Mountain View, CA).

Optical images observed (and recorded, if necessary) by a camera or other recording device (eg, photodiodes and data storage) are optionally further optional in this specification. Is processed (eg, by digitizing the image and / or storing and analyzing the image on a computer). Various commercial peripherals and software include, for example, PC (DOS).TM, OSTM WINDOWS®, WINDOWS® NTTMOr WINDOWS® 95TMCompatible with machines based on Intel x86 or Pentium® chips), MACINTOSHTM, Or UNIX® based (eg SUN)TMIt is available for storage and analysis of digitized, digitized video or digitized optical images using a computer (workstation).

One conventional system transfers light from an assay device to a cooled charge-coupled device (CCD) camera and is commonly used in the art. A CCD camera includes an array of pixels. The light from the subject is imaged on the CCD. Specific pixels corresponding to a region of the subject (eg, individual hybridization sites on an array of biological polymers) are sampled to obtain a light intensity reading for each position. Multiple pixels are processed as the speed increases. The devices and methods of the present invention are readily used, for example, for observing any sample by fluorescence or darkfield microscopy techniques.

(Other polynucleotide compositions) The invention also includes compositions comprising two or more polynucleotides of the invention, such as substances for recombination. The composition may comprise a library of recombinant nucleic acids, wherein the library comprises at least 2, 3, 5, 10, 20, or 50 or more polynucleotides. This polynucleotide is optionally cloned into an expression vector that provides an expression library.

The invention also uses the restriction endonuclease RNAse, or DNAse, to digest one or more polynucleotides of the invention (eg, as performed in the particular recombinant modalities described above). The compositions produced; and the compositions produced by fragmenting or shearing one or more polynucleotides of the invention by mechanical means (eg, ultrasound treatment, vortex, etc.). Can also be used to provide a substance for recombination in the above methods. Similarly, a composition comprising a set of oligonucleotides corresponding to more than one nucleic acid of the invention is useful as a recombinant and is a feature of the invention. For convenience, these fragmented mixtures, sheared mixtures, or oridnucleotide-synthesized mixtures are referred to as fragmented nucleic acid sets.

Also included in the invention are compositions produced by incubating one or more fragmented nucleic acid sets in the presence of ribonucleotide triphosphate or deoxyribonucleotide triphosphate and nucleic acid polymerase. The resulting composition forms a recombinant mixture for many of the recombinant modes described above. This nucleic acid polymerase can be an RNA polymerase, a DNA polymerase, or an RNA-directed DNA polymerase (eg, "reverse transcriptase"); this polymerase can be, for example, a thermostable DNA polymerase (eg, VENT, TAQ, etc.). possible. (Integrated system) The present invention provides, for example, sequence information herein for polypeptides and nucleic acids herein, including these sequences listed herein, and various silent and conservative substitutions thereof. Provided are computers containing strings corresponding to, computer-readable media, and integrated systems.

For example, various methods and genetic algorithms (GAs) known in the art can be used to detect homology or similarity between different strings, or other desired functions (eg, output). It can be used to perform (controlling files) and provides the basis for creating presentations of information, including arrays. An example is BLAST (discussed above).

Thus, different types of homology and similarity with varying stringencies and lengths can be detected and recognized within the integrated system herein. For example, many homology determination methods have been designed for comparative analysis of biopolymer sequences, spell checking in word processing, and retrieval of data from various databases. Models that simulate string annealing of complementary homologous polynucleotides using an understanding of the complementary interactions of double helix pairing between the four major nucleic acid bases in a native polynucleotide are also described herein. Used as the basis for array alignment or other operations typically performed on the string corresponding to the array in (eg, word processing operations, building diagrams containing strings of arrays or subarrays, output tables, etc.) Can be. An example of a software package using GA for calculating sequence similarity is BLAST, which can be applied to the present invention by entering the string corresponding to the sequence in the present specification.

Similarly, standard desktop applications such as word processing software (eg Microsoft Word)TMOr Corel WordPerfectTM) And database software (eg spreadsheet software) and database software (eg Microsoft Excel)TM, Corel Quattro ProTM) Or database program (for example, Microsoft Access)TMOr ParadoxTM)) Can be adapted to the present invention by entering a string corresponding to the GAT homolog of the present invention (nucleic acid, protein, or both). For example, this integrated system works with a user interface (eg, a GUI in a standard operating system such as a Windows® system, Macintosh system, or LINUX system) to manipulate a string of characters. It may include the aforementioned software with appropriate string information used. As described, specialized alignment programs such as BLAST can also be incorporated into the system of the invention for the alignment of nucleic acids or proteins (or corresponding strings).

An integrated system for analysis in the present invention typically includes a digital computer with GA software for aligning sequences, and a dataset that is input to a software system that includes any of the sequences herein. .. This computer is, for example, a DOS compatible with a PC (Intel x86 or Pentium® chip).TM, OS2TM, WINDOWS (registered trademark), WINDOWS (registered trademark) NT, WINDOWS (registered trademark) 95, WINDOWS (registered trademark) 98, LINUX-based machine, MACINTOSHTM, Power PC or UNIX® (eg SUNTMIt can be a workstation) based machine, or any other commercially common computer known to those of skill in the art. Software for aligning arrays, or software for manipulating arrays, is available or standard programming languages (eg Visualbasic, Fortran, Basic, Java®, etc.) It can be easily constructed by those skilled in the art.

Any controller or computer optionally includes a monitor, which is often a cathode ray tube (CRT) display, a flat panel display (eg, an active matrix liquid crystal display, a liquid crystal display), and the like. Computer circuits are often placed in boxes that contain a large number of integrated circuit chips (eg, microprocessors, memory, interface circuits, etc.). The box also includes hard disk drives, floppy (registered trademark) disk drives, high capacity removable drives (eg, writable CD-ROMs), and other common peripherals, as needed. The input device (eg, keyboard or mouse) provides user input from the user and user selection of arrays that are compared or otherwise manipulated in the associated computer system, as needed.

The computer typically enters in any of the user forms entered into the set parameter area (eg, in the GUI or pre-programmed instructions (eg, pre-programmed instructions for a variety of different specific operations). ) Includes appropriate software to receive user instructions. The software then translates these instructions into the appropriate language for commanding the flow direction and the operation of the transport controller in order to perform the desired operation.

The software also uses an output element to control nucleic acid synthesis (eg, based on sequences or sequence alignments herein), or alignments performed using strings corresponding to the sequences herein. It may include other operations that occur downstream of the other operations. Thus, the nucleic acid synthesizer can be a component in one or more integrated systems herein.

In a further aspect, the invention provides a kit that embodies the methods, compositions, systems and devices herein. The kits of the invention optionally include one or more of: (1) devices, systems, system components, or device components as described herein; (2) the present. Instructions for performing the methods described herein, and / or instructions for manipulating the apparatus or components of the apparatus herein, and / or using the compositions herein. Instructions; (3) one or more GAT compositions or components; (4) components or containers for holding the compositions, and (5) packaging material.

In a further aspect, the invention describes the use of any device, component, composition, or kit herein, performing any method or assay herein, and / or herein. Provided is the use of any device or kit for performing any assay or method of.

(Host cells and organisms) The host cell can be a eukaryote (eg, a eukaryote, a plant cell, an animal cell, a protoplast, or a tissue culture). The host cell contains a plurality of (for example) cells (for example, an organism) as required. Alternatively, the host cell can be bacteria (ie, gram-positive bacteria, red-colored bacteria, green sulfur bacteria, green non-sulfur bacteria, cyanobacteria, spiroheta, thermotogales, flavobacterium, and bacteroids) and primordial bacteria (ie, Col Paleozoic). Bacteria, Termoproteus, Pyrodictium, Termocox, Methanbacteria, Alcaeglobus and highly eophilic bacteria), but are not limited to protozoa.

A transgenic plant or plant cell incorporating the GAT nucleic acid of the present invention and / or a transgenic plant or plant cell expressing the GAT polypeptide of the present invention is a feature of the present invention. Transformation of plant cells and protoplasts can be performed by essentially any variety of methods known to those skilled in the art of plant molecular biology, including but not limited to those described herein. .. See Methods in Enzymology, Vol.153 (Recombinant DNA Part D) Wu and Grossman (eds.) 1987, Academic Press, which are generally incorporated herein by reference. As used herein, the term "transformation" means a change in the genotype of a host plant upon introduction of a nucleic acid sequence (eg, a "heterologous" or "foreign" nucleic acid sequence). This heterologous nucleic acid sequence does not necessarily have to come from a different source, but in some respects the heterologous nucleic acid sequence is lateral to the cell into which it is introduced.

In addition to Berger, Ausubel and Shamblook, as a useful general reference for cloning, culturing and regenerating plant cells, Jones (eds.) (1995) Plant Gene Transfer and Expression Protocols-Methods in Molecular Biology, Volume 49 Humana Press Towata NJ Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY (Payne); and Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New) York) (Gamborg). Various cell culture media are described in Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca, Raton, FL (Atlas). Further information on plant cell culture is available in the commercial literature (eg, Life Science Research Cell Culture Catalog (1998) (Sigma-LSRCCC) from Sigma-Aldrich, Inc (St Louis, MO), and also, for example, Sigma. -Found in the Plant Culture Catalog from Aldrich, Inc (St Louis, MO) and Addendum (1997) (Sigma-PCCS)). Further details on plant cell culture can be found in Croy (eds.) (1993) Plant Molecular Biology Bios Scientific Publishers, Oxford, UK).

In one embodiment of the invention, a recombinant vector containing one or more GAT polynucleotides suitable for transformation of plant cells is prepared. The DNA sequence encoding the desired GAT polypeptide (selected from, for example, SEQ ID NOs: 1-5 and 11-262) is convenient for constructing a recombinant expression cassette that can be introduced into the desired plant. used. In the context of the present invention, the expression cassette is typically a promoter sequence sufficient to direct transcription of the GAT sequence in the intended tissue of the transformed plant (eg, whole plant, leaves, roots, etc.). Includes selected GAT polynucleotides that are operably linked to other transcriptional and translational initiation regulatory sequences.

For example, a strong constitutive plant promoter or a weak constitutive plant promoter that directs GAT nucleic acid expression in all plant tissues can be conveniently utilized. Such promoters are active under most environmental conditions and under developmental or cell differentiation conditions. Examples of constitutive promoters include the 1'-promoter or 2'-promoter of Agrobacterium tumefaciens, and other transcription initiation regions derived from various plant genes known to those of skill in the art. If overexpression of the GAT polypeptide of the invention is detrimental to plants, one of ordinary skill in the art will recognize that a weak constitutive promoter can be used for low levels of expression. In these cases, if high levels of expression are not harmful to the plant, a strong promoter (eg, t-RNA, or other pol III promoter, or a strong pol II promoter (eg, cauliflower mosaic virus promoter, CaMV, 35S)). Promoter)) can be used.

Alternatively, the plant promoter can be under environmental control. Such promoters are referred to as "inducible" promoters. Examples of environmental conditions in which transcription can be altered by an inducible promoter include pathogen attack, anaerobic conditions, or the presence of light. In some cases, it is desirable to use promoters that are "tissue-specific" and / or under developmental control, so that this GAT polynucleotide is present in a particular tissue or developmental stage (eg, leaves, roots, etc.). It is expressed only in seedlings, etc.). Endogenous promoters of genes for herbicide resistance and related phenotypes are GAT nucleic acids (eg, P450 monooxygenase, glutathione-S-transferase, homoglutathione-S-transferase, glyphosate xidase, and 5-enolpyrvir simisonic acid. It is particularly useful for promoting the expression of -2-phosphate synthase).

Tissue-specific promoters can also be used to direct the expression of heterologous structural genes, including the GAT polynucleotides described herein. Thus, this promoter is any gene whose expression is desired in the transgenic plants of the invention (eg, GAT and / or other genes that confer herbicide resistance or herbicide resistance, other useful properties (eg, eg). , Can be used in recombinant expression cassettes that drive the expression of genes that affect heterosis). Similarly, enhancer elements (eg, derived from 5'regulatory sequences, or introns of heterologous genes) can also be used to enhance expression of heterologous structural genes such as GAT polynucleotides.

In general, the particular promoter used in a plant expression cassette depends on the intended application. Any number of promoters that direct transcription in plant cells may be suitable. This promoter can be either constitutive or inducible. In addition to the promoters described above, bacterial promoters engineered in plants include the Octopin synthase promoter, the Noparin synthase promoter, and other promoters derived from the Ti plasmid. See Herrera-Estrella et al. (1983) Nature 303: 209. Virus promoters include CaMV's 35SRNA promoter and 19SRNA promoter. See Odell et al. (1985) Nature 313: 810. Other plant promoters include the small subunit promoter of ribulose-1,3-diphosphate carboxylase, and the phaseolin promoter. E8 gene (Deikman and Fischer (1988) EMBO J Promoter sequences from 7: 3315) and other genes are also conveniently used. Promoters specific for monocotyledonous plant species are also considered (McElroy D., Brettell RIS 1994. Foreign gene expression in transgenic cereals. Trends Biotech., 12: 62-68). Alternatively, novel promoters with useful features can be identified from any viral, bacterial or plant source by methods known in the art, including sequence analysis, enhancer trapping or promoter trapping.

In the preparation of the expression vector of the present invention, sequences other than the promoter and the gene encoding GAT are also conveniently used. If appropriate polypeptide expression is desired, the polyadenylation region can be derived from a natural gene, various other plant genes, or T-DNA. Signal / localized peptides (eg, which promote the migration or extracellular secretion of expressed polypeptides to internal organelles (eg, chloroplasts)) can also be used.

Vectors containing GAT polynucleotides may also contain marker genes that give plant cells a selectable phenotype. For example, this marker encodes killing agent resistance, especially antibiotic resistance (eg, resistance to kanamycin, G418, bleomycin, hygromycin) or herbicide resistance (eg, resistance to chlorosulfurone, or phophinothricin). obtain. Via visible reaction products (eg, β-glucuronidase, β-galactosidase, and chloramphenicol acetyltransferase) or the gene product itself (eg, green fluorescent protein, GFP; Sheen et al., (1995) The Plant Reporter genes used to monitor gene expression and protein localization through direct visualization of Journal 8: 777) are used, for example, to monitor transient gene expression in plant cells. Can be. Transient expression systems can be used, for example, in plant cells when screening plant cell cultures for herbicide resistance activity.

(Plant transformation) (protoplast) Numerous protocols for establishing transformable protoplasts from various plant types, followed by transformation of cultured protoplasts, are available in the art and are incorporated herein by reference. For example, Hashimoto et al. (1990) Plant Physiol. 93: 857; Fowke and Constabel (ed.) (1994) Plant Protoplasts; Saunders et al. (1993) Applications of Plant In Vitro Technology Symposium, UPM 16-18; and Lyznik et al. (1991). ) See BioTechniques 10: 295, each of which is incorporated herein by reference.

(chloroplast) Chloroplasts are the site of action of some herbicide resistance activities, and in some examples, this GAT polynucleotide is chloroplasts to facilitate the transfer of gene products into chloroplasts. It is fused to a body transport sequence peptide. In these cases, it may be convenient to transform the GAT polynucleotide into the chloroplast of the plant host cell. Numerous methods are available in the art to achieve chloroplast transformation and expression (eg, Daniell et al. (1998) Nature Biotechnology 16: 346; O'Neill et al. (1993) The Plant Journal 3). : 729; Maliga (1993) TIBTECH 11: 1). This expression construct comprises a functional transcriptional regulatory sequence in a plant operably linked to a polynucleotide encoding a GAT polypeptide. Expression cassettes designed to function in chloroplasts (eg, expression cassettes containing GAT polynucleotides) contain the sequences necessary to ensure expression in chloroplasts. Typically, two regions homologous to the chloroplast genome flank the coding sequence to result in homologous recombination using the chloroplast genome; often, selectable marker genes also arise. (transplastonic) Present in adjacent plastid DNA sequences to facilitate selection of genetically stable transformed chloroplasts in plant cells (eg, Maliga (1993) and Daniell (1998), and these. See references cited in).

(General transformation method) The DNA constructs of the present invention can be introduced into the desired plant host genome by a variety of conventional techniques. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, for example, Payne, Gamborg, Croy, Jones et al., All Supra, and, for example, Weising et al. (1988) Ann. Rev. Genet. 22: 421.

For example, DNA can be introduced directly into plant cell genomic DNA using techniques (eg, plant cell protoplast electroporation and microinjection), or DNA constructs are ballistic, such as DNA particle bombardment. It can be introduced directly into plant tissue using the method. Alternatively, the DNA construct can be bound to the appropriate T-DNA proximity region and introduced into a conventional Agrobacterium tumefaciens host vector. The toxic function of the Agrobacterium host directs the insertion of constructs and nearby markers into plant cell DNA when plant cells are infected by bacteria.

Microinjection techniques are well known in the art and are well documented in scientific and patent literature. Introduction of DNA constructs using polyethylene glycol precipitates is described in Paszkowski et al. (1984) EMBO J 3:2717. Electroporation techniques are described in Fromm et al. (1985) Proc Nat'l Acad Sci USA 85: 5824. Ballistic transformation techniques are described in Klein et al. (1987) Nature 327: 70; and Weeks et al. Plant Physiol 102: 1077.

In some embodiments, Agrobacterium-mediated transformation techniques are used to transfer the GAT sequences of the invention to transgenic plants. Although Agrobacterium-mediated transformation is widely used for transformation of dicotyledonous plants, certain monocotyledonous plants can also be transformed by Agrobacterium. For example, Agrobacterium transformation of rice was carried out by Hiei et al. (1994) Plant J. 6: 271; U.S. Pat. No. 5,187,073; U.S. Pat. No. 5,591,616; Li et al. (1991) Science in China 34:54; and Raineri et al. (1990). Described by Bio / Technology 8:33. Maize, barley, rye and asparagus transformed by Agrobacterium-mediated transformation have also been described (Xu et al. (1990) Chinese J Bot 2:81).

Agrobacterium-mediated transformation techniques utilize the ability of A. tumefaciens' tumor-inducing (Ti) plasmid to integrate into the plant cell genome and simultaneously transfer the nucleic acid of interest into plant cells. Typically, an expression vector is generated, where the nucleic acid of interest (eg, the GAT polynucleotide of the invention) is attached to a self-replicating plasmid that also contains the T-DNA sequence. The T-DNA sequence typically flanks the expression cassette nucleic acid of interest and contains the plasmid integration sequence. In addition to the expression cassette, T-DNA also typically contains marker sequences (eg, antibiotic resistance genes). The plasmid with T-DNA and expression cassette is then transfected into Agrobacterium cells. Typically, for effective transformation of plant cells, A. tumefaciens bacteria also possess the required vir region integrated on or on their chromosomes. Consideration of Agrobacterium-mediated transformation, Firoozabady and Kuehnle (1995) Plant Cell Tissue and Organ Culture See Fundamental Methods, Gamborg and Phillips.

(Regeneration of transgenic plants) Transformed plant cells obtained by plant transformation techniques including the above techniques are acquired for the transformed genotype (ie, GAT polynucleotide) and thus the desired phenotype (glyphosate or glyphosate analog). It can be cultivated to regenerate an entire plant with resistance (ie, resistance). Such regeneration techniques depend on the manipulation of specific plant hormones in tissue culture growth medium and typically depend on the killing and / or herbicide markers introduced with the desired nucleotide sequence. .. Alternatively, selections for the glyphosate resistance conferred by the GAT polynucleotides of the invention can be performed. Plant regeneration from cultured protoplasts is described by Evans et al. (1983) Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp 124-176, Macmillan Publishing Company, New York; and Binding (1985) Regeneration of Described in Plants, Plant Protoplasts pp 21-73, CRC Press, Boca Raton. Regenerated products can also be obtained from plant callus, plant explants, plant organs, or parts thereof. Such regeneration techniques are generally described in Klee et al. (1987) Ann Rev of Plant Phys 38: 467. See also Payne and Gamborg, for example. After transformation with Agrobacterium, the explants are typically transferred to selective medium. Those skilled in the art will appreciate that the selective medium depends on a selectable marker that is simultaneously transfected into the explants. After a suitable length of time, the transformant begins to form buds. After the shoots are about 1-2 cm long, the shoots should be transferred to a suitable root and shoot medium. Selective pressure should be maintained in root and shoot medium.

Typically, this transformant develops roots and forms small plants in about 1-2 weeks. After the small plants reach a height of about 3-5 cm, they are transferred to the subsoil soil in the fiber pot. Those skilled in the art will appreciate that different acclimatization procedures are used to obtain transformed plants of different species. For example, after root and shoot development, the cut and transformed somatic embryos of the plant are transferred to the medium for the establishment of small plants. See, for example, Dodds and Roberts (1995) Experiments in Plant Tissue Culture, Volume 3, Cambridge University Press for a description of the selection and regeneration of transformed plants.

Vacuum infiltration (Bechtold N., Ellis J. and Pelletier G., 1993, In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants.CR Acad Sci Paris Life Sci 316: 1194-1199) and simple flowering plants Immersion (Desfeux, C., Clough SJ, and Bent AF, 2000, Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method.Plant There are also methods for Agrobacterium transformation of Arabidopsis using Physiol. 123: 895-904). Using these methods, transgenic seeds are produced without the need for tissue culture.

There are plant variants for which efficient Agrobacterium-mediated transformation protocols are still being developed. For example, successful tissue transformation, which produces transgenic plants in conjunction with transformed tissue regeneration, has not been reported for some of the most commercially relevant and cotton varieties. Nonetheless, the approaches that can be used with these plants are the step of stably introducing this polynucleotide into the varieties of related plants via Agrobacterium-mediated transformation, the step of confirming operability, and then the standard. It comprises the step of transferring this transgene to a desired commercial lineage using a conventional sexual or backcrossing technique. For example, in the case of cotton, Agrobacterium can be used to transform the Cooker line of Gossypium hirustum (eg, Cooker line 310, 312, 5100 Deltapine 61 or Stoneville 213), and then this transgene is separated by backcrossing. Can be introduced into the more commercially relevant G. hirustum varieties.

The transgenic plants of the invention can be characterized either genotypically or phenotypically to determine the presence of the GAT polynucleotides of the invention. Genotyping can be performed by any of many well-known techniques, including PCR amplification of genomic DNA and hybridization of genomic DNA with specific labeled probes. Phenotypic analysis includes, for example, survival of plants or plant tissues exposed to selected herbicides such as glyphosate.

Essentially any plant can be transformed with the GAT polynucleotides of the invention. Suitable plants for the transformation and expression of novel GAT polynucleotides of the invention include agronomically and horticulturally important species. Such species include, but are not limited to, members of the following families: Gramineae (including corn, lime, rye wheat, barley, millet, rice, wheat, Rosaceae, etc.); Leguminosae (legumes) , Beans, lens beans, peanuts, kuzuimo, scorpionfish, hassho beans, soybeans, clover, alfalfa, rubinus, vetch, hass, sweet clover, wisteria, and sweet pea); The largest family of legumes (including important commercial crops such as sunflowers) and Rosaceae (including raspberries, apricots, almonds, peaches, roses, etc.) and nut plants (including walnuts, pecans, hazelnuts, etc.) and forests. Trees (including Pinus, Quercus, Pseutotsuga, Sequoia, Populus, etc.).

Agrostis, Allium, Antirrhinum, Apium, Arachis, Asparagus, Atropa, Avena (eg bats), Bambusa, Brassica, Bromus, Browaalia, Camellia, Cannabis, Capsicum, Cicer, Chenopodium, Chichorium, Citers, Coffea, Coix, Cu , Cynodon, Dactylis, Datura, Daucus, Digitalis, Dioscorea, Elaeis, Eleusine, Festuca, Fragaria, Geranium, Gossypium, Glycine, Helianthus, Heterocallis, Hevea, Hordeum (eg barley), Hyoscyamus, Ipomoea Linum, Lolium, Lotus, Lycopersicon, Majorana, Malus, Mangifera, Manihot, Medigo, Nemesia, Nicotiana, Onobrychis, Oryza (eg rice), Panicum, Pelargonium, Pennisetum (eg millet), Petunia, Pisum, Phaseol Poa, Prunus, Ranunculus, Raphanus, Ribes, Ricinus, Rubus, Saccharum, Salpiglossis, Secrete (eg rye), Senecio, Setaria, Sinapis, Solanum, Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonella, Triticum (eg wheat) Vicia, Vigna, Vitis, Zea (eg corn), as well as Olyreae, Pharoideae and many other genera. Gram as described

Common crop plants targeted by the present invention include corn, rice, rye, rye, cotton, soybean, sugar cane, small wheat, crow wheat, large wheat, millet, sunflower, abrana, peas, beans, and lens beans. , Peanuts, kuzuimo, sage, velvet beans, clover, alfalfa, rubinus, vetch, hass, sweet clover, wisteria, sweet pea, and nut plants (eg, walnuts, pecans, etc.).

In one aspect, the present invention is resistant to glyphosate as a result of being transformed with a gene encoding glyphosate N-acetyltransferase under conditions such that the crop plant produces and collects the crop. Provided is a method for producing a crop by growing a certain crop plant. Preferably, glyphosate is applied to the plant or its close relatives at a concentration effective in controlling weeds without the transgenic crop plant impairing crop growth and production. Glyphosate can be applied before cultivation or at any time after harvest, including until and at harvest. Glyphosate can be applied once or multiple times. The timing of glyphosate application, the amount applied, the mode of application, and other parameters will vary based on the particular nature and habitat of the crop plant and can be readily determined by one of ordinary skill in the art. The present invention further provides the growth of crops produced by the method.

The present invention provides the growth of plants containing the GAT polynucleotide transgene. This plant can be, for example, a monocotyledonous plant or a dicotyledonous plant. In one aspect, proliferation necessarily involves crossing a plant containing the GAT polynucleotide transgene with a second plant, so that at least some offspring of the cross show glyphosate resistance.

In one aspect, the invention provides a method for selectively controlling weeds in the field in which the crop is grown. This method grows glyphosate-tolerant crop seeds or crop plants as a result of being transformed with a gene encoding GAT (eg, a GAT polynucleotide), and has significant detrimental effects on the crop. Includes applying sufficient amounts of glyphosate to crops and any weeds to control weeds without them. The crop need not be totally insensitive to this herbicide, as long as the benefits derived from weed inhibition are more important than the negative effects of glyphosate or glyphosate analogs on either the crop or the crop plant. It is important to note that.

In another aspect, the invention provides the use of GAT polynucleotides as selectable marker genes. In this embodiment of the invention, the presence of a GAT polynucleotide in a cell or organism confers on the cell or organism a detectable glyphosate-resistant phenotypic trait, thereby linking to the GAT polynucleotide of interest. It can be selected for genetically transformed cells or organisms. Thus, for example, a GAT polynucleotide can be introduced into a nucleic acid construct (eg, a vector), whereby host proliferation in the presence of glyphosate, as well as a host lacking the nucleic acid construct, survives or proliferates. Allows identification of hosts containing nucleic acid constructs (eg, cells or transgenic plants) by selection for their ability to survive and / or proliferate at discriminatingly fast rates. GAT polynucleotides have been used as selectable markers in a wide variety of glyphosate-sensitive hosts, including plants, most bacteria (including E. coli), actinomycetes, yeasts, algae, and fungi. obtain. One advantage of using herbicide-resistant strains as markers in plants over traditional antibiotic-resistant strains is that it is a concern for some members of the application that antibiotic-resistant strains escape into the environment. Is to avoid. Some experimental data from experiments demonstrating the use of GAT polynucleotides as selectable markers in various host systems are described in the Examples section herein.

(Selection of gat polynucleotides that confer enhanced glyphosate resistance in transgenic plants) A library of GAT-encoding nucleic acids diversified according to the methods described herein can be selected for their ability to confer resistance to glyphosate in transgenic plants. After one or more cycles of diversification and selection, the modified GAT gene is used as a selectable marker that facilitates the production and evaluation of transgenic plants, as well as a method of conferring herbicide resistance on experimental or agricultural plants. obtain. For example, after diversification of any one or more of SEQ ID NOs: 1 to 5 to produce a library of diversified GAT polynucleotides, the first functional assessment is E. coli. It can be performed by expression of the GAT coding sequence library in. The expressed GAT polypeptide can be purified as described above or partially purified and screened for improved rates by mass spectrometry. After one or more rounds of diversification and selection, the polynucleotide encoding the improved GAT polypeptide, for example, is a potent constitutive promoter (eg, CaMV). It is cloned into a plant expression vector operably linked to the 35S promoter). This expression vector containing modified GAT nucleic acid is typically by Agrobacterium-mediated transformation of Arabidopsis. Transformed into a thaliana host plant. For example, Arabidopsis hosts can be easily transformed by immersing the inflorescences in a solution of Agrobacterium and allowing them to grow and seed. Thousands of seeds recover in about 6 weeks. These seeds are then collected in large quantities from the soaked plants and germinate in the soil. In this mode, thousands of independently transformed plants that make up the high throughput (HTP) plant transformation form can be produced for evaluation. Large numbers of grown seedlings are sprayed with glyphosate, and surviving seedlings exhibiting glyphosate resistance survive in the selection process, while non-transgenic plants and plants incorporating less preferably modified GAT nucleic acids Damaged or killed by herbicide treatment. If desired, GAT-encoding nucleic acids that provide improved resistance to glyphosate are recovered, for example, by PCR amplification with T-DNA primers flanking the library insert, and in further diversification procedures or homologous. Alternatively, it is used to produce additional transgenic plants of different species. If desired, further round diversification and selection can be accomplished by using increasing the concentration of glyphosate in each contiguous compartment. In this mode, GAT polynucleotides and polypeptides are obtained that impart resistance to glyphosate concentrations useful in field conditions.

(Herbicide resistance) The mechanism of glyphosate resistance of the present invention can be combined with other forms of glyphosate resistance known in the art for producing plants and plant explants with excellent glyphosate resistance. For example, inserting a glyphosate-tolerant plant into its genome with the ability to produce high levels of 5-enolpyrvir simimate-3-phosphate synthase (EPSP), as described more fully by: Can be produced by: U.S. Pat. Nos. 6,248,876 B1; U.S. Pat. No. 5,627,061; No. 5,804,425; No. 5,633,435; No. 5,145,783; No. 4,971,908; No. 5,312,910; No. 5,188,642; No. 4,940,835. No. 5,866,775; No. 6,225,114 B1; No. 6,130,366; No. 5,310,667; No. 4,535,060; No. 4,769,061; No. 5,633,448; No. 5,510,471; No. 36,449 No. 37,287 E; and US Pat. No. 5,491,288; and International Application WO 97/04103; WO 00/66746; WO 01/66704; and WO 00/66747 (these are for all purposes throughout the invention. It is used as a reference for this purpose.) Glyphosate resistance is also more fully described in US Pat. Nos. 5,776,760 and 5,463,175, which are incorporated herein by reference in their entirety for all purposes. It can also be transmitted to plants that express the gene encoding the enzyme.

In addition, the glyphosate resistance mechanism of the present invention can be combined with other modes of herbicide resistance to produce plants and plant explants that are resistant to glyphosate and one or more other herbicides. For example, hydroxyphenylpyrvate dioxygenase is an enzyme that catalyzes the conversion of para-hydroxyphenylpyrvate (HPP) into homogenic acid. Molecules that bind to this enzyme to inhibit this enzyme and to inhibit the conversion of HPP to homogenic acid are useful as herbicides. Plants that are more resistant to certain herbicides are described in U.S. Pat. Nos. 6,245,968 B1; 6,268,549; and 6,069,115; and International Application WO99 / 23886 (these are hereby for all purposes). Incorporated throughout).

Sulfonyl urea and imidazolinone herbicides also inhibit higher plant growth by blocking acetolactate synthase (ALS) or acetolactate synthase (AHAS). The production of sulfonylurea and imidazolinone-resistant plants is described in US Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107. No. 5,928,937; and 5,378,874; and International Application WO 96/33270, which are incorporated herein by reference for all purposes. ..

Glutamine synthetase (GS) has been shown to be an essential enzyme required for the development and survival of most plant cells. Inhibitors of GS are toxic to plant cells. Glufosinate herbicides have been developed based on the toxic effects resulting from the inhibition of GS in plants. These herbicides are non-selective. They inhibit the growth of all the different species of plants present and cause the destruction of all of them. The development of plants containing exogenous phosphinosricin acetyltransferase has been developed in US Pat. Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477. It is described in No. 5,646,024; No. 6,177,616 B1; and No. 5,879,903 (these are incorporated herein by reference in its entirety for all purposes).

Protoporphyrinogen oxidase (protox) is required for the production of chlorophyll, which is necessary for the survival of all plants. This protox enzyme acts as a target for various herbicide compounds. These herbicides also inhibit the growth of all the different species of plants present and cause the destruction of all of them. The development of plants with altered protox activity that is resistant to these herbicides has been developed in US Pat. Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373; and International Application WO01 / 12825 (these). Is incorporated herein by reference in its entirety for all purposes).

(Example) The following examples are exemplary and not limiting. One of ordinary skill in the art will recognize various non-limit parameters that can be varied to achieve essentially similar results.

(Example 1: Isolation of a novel natural GAT polynucleotide) Five native GAT polynucleotides (ie, naturally occurring GAT polynucleotides in non-genetically modified organisms) were found by expression cloning of sequences from the Bacillus lineage exhibiting GAT activity. These nucleotide sequences were determined and provided herein as SEQ ID NOs: 1-5. That is, a collection of about 500 Bacillus and Pseudomonas strains was screened for their natural ability to N-acetylate glyphosate. These lines were grown overnight in LB, collected by centrifugation, permeable in dilute toluene, then washed and resuspended in a reaction mixture containing buffer, 5 mM glyphosate, and 200 μM acetyl-CoA. It became cloudy. The cells were incubated in the reaction mixture for 1-48 hours (during this time period, the same volume of methanol was added to the reaction). The cells are then pelleted by centrifugation and the supernatant is parent ion mode mass. Filtered before analysis by spectroscopy). The reaction product was positively identified as N-acetylglycosate by comparing the mass spectrometric profile of the reaction mixture with the standard N-acetylglyphosate shown in FIG. Product detection was dependent on both substrate (acetyl-CoA and glyphosate) inclusions and was disrupted by thermal denaturation of bacterial cells.

Individual GAT polynucleotides were then cloned from strains identified by functional screening. Genomic DNA was prepared and partially digested with the Sau3A1 enzyme. A fragment of about 4 Kb was cloned into an E. coli expression vector and transformed into an electrocompetent E. coli. Individual clones exhibiting GAT activity were identified by post-reaction mass spectrometry as previously described, except that toluene washing was replaced by permeation with PMBS. Genomic fragments were sequenced and the open reading frame encoding the putative GAT polypeptide was identified. Identification of the GAT gene was confirmed by expression of an open reading frame in E. coli and detection of high levels of N-acetylglycosphate produced from the reaction mixture.

(Example 2: characterization of GAT polypeptide isolated from B. licheniformis line B6) Genomic DNA from B. licheniformis line B6 was purified, partially digested with Sau3A1, and fragments of 1-10 Kb were cloned into E. coli expression vectors. A clone with a 2.5 kb insert gave glyphosate N-acetyltransferase (GAT) activity on an E. coli host as determined using mass spectral analysis. Sequencing of the insert revealed a single complete open reading frame of 441 base pairs. Subsequent cloning of this open reading frame confirmed that it encodes the GAT enzyme. A plasmid (pMAXY2120, shown in Figure 4) carrying the gene encoding the B6 GAT enzyme was presented in E. coli strain XL1. Transformed to Blue. A saturated culture of 10% innoculum was added to the Lurias (broth) and the culture was incubated at 37 ° C for 1 hour. GAT expression was induced by the addition of IPTG at a concentration of 1 mM. The culture was incubated for an additional 4 hours, after which the cells were collected by centrifugation and the cell pellet was stored at -80 ° C.

Cell lysis was provided by adding 1 ml of the following buffer to 0.2 g of cells: 25 mM HEPES (pH 7.3), 100 mM KCl with 0.1 mM EDTA and 10% methanol (HKM). ), 1 mM DTT, 1 mg / ml chicken egg lysozyme, and a prosthesis inhibitor buffer obtained from Sigma and used according to the manufacturer's instructions. After 20 minutes of incubation at room temperature (eg 22-25 ° C), dissolution was achieved with simple sonication. The lysate was centrifuged and the supernatant was equilibrated with HKM for Sephadex. Desalted through G25. A partially purified product was obtained by affinity chromatography on CoA agarose (Sigma). The column was equilibrated with HKM and the clarified extract was passed under hydrostatic pressure. Unbound proteins were removed by washing the column with HKM and GAT was eluted with HKM containing 1 mM Coenzyme A. This procedure provided a 4-fold purified product. At this stage, about 65% of protein staining observed on SDS polyacrylamide gels filled with crude lysates is due to GAT, and another 20% is vector-encoded chloramphenicol acetyltransferase. Due to.

Purification for homogeneity was obtained by gel filtration of the partially purified protein via Superdex 75 (Pharmacia). The moving layer was HKM, where GAT activity was eluted in a volume corresponding to the molecular radius of 17 kD. This material was as uniform as judged by the Kumashi stain of 3 μg GAT samples subjected to SDS polyacrylamide gel electrophoresis on a 12% acrylamide gel (1 mm thick). Purification achieved a 6-fold increase in specific activity.

Glyphosate's apparent KMA reaction mixture containing 5 mM morpholine, saturated (200 μM) acetyl-CoA, various concentrations of glyphosate, and 1 μM purified GAT in a buffer prepared to pH 7.7 with acetic acid and 20% ethylene glycol. Determined above. The initial reaction rate was determined by continuously monitoring the hydrolysis of the thioester bond of acetyl-CoA at 235 nm (E = 3.4 OD / mM / cm). Observe the saturated hyperbolic kinetics (Fig. 5), from which the apparent K of 2.9 ± 0.2 (SD) mMMGot

Apparent K of AcCoAMWas determined on a reaction mixture containing 5 mM morpholine, prepared to pH 7.7 with acetic acid and 5 mM glyphosate, various concentrations of acetyl-CoA, and 0.19 μM GAT in 50% methanol. The initial reaction rate was determined using mass spectrometric detection of N-acetylglyphosate. 5 μl was repeatedly injected into the instrument and reaction rates were obtained by plotting reaction time vs. integrated peak area (Figure 6). Saturated hyperbolic kinetics are observed (Fig. 7), from which the apparent K of 2 μMMLed. From the value of Vmax obtained from the enzyme of known concentration, kcat of 6 / min was calculated.

(Example 3: Mass Spectrometry (MS) Screening Process) Samples (5 μl) were aspirated from a 96-well microtiter plate at a rate of 1 sample every 26 seconds and injected into a triple quadrupole mass spectrometer (Micromass Quattro LC, triple quadrupole mass spectrometer) without any separation. The sample was transferred to a mass spectrometer by a moving layer of water / methanol (50:50) at a flow rate of 500 Ul / min. Each injected sample is subjected to a negative electrospray ionization process (needle voltage, -3.5KV; cone voltage, 20V; source temperature 120C; desolvation temperature, 250C; Ionized by cone gas flow rate, 90 L / hour; and non-solvate gas flow rate, 600 L / hour). This molecular ion (m / z 210) formed during this process is transferred to the second quadrupole (pressure here, 5 × 10).-4Selected by the first quadrupole to perform collision induced dissociation (CID) at mBar and collision energy adjusted to 20 Ev). At the third quadrupole, one of the daughter ions (m / z 124) produced from the parent ion (m / z 210) reaches the detector for signal recording. Installed only for what can be done. A first quadrupole and a third quadrupole were installed in the resolution unit (where the photomultiplier was operated at 650 V). Pure standard N-acetylglyphosate was used for comparison and integrated peaks were used to estimate the concentration. By this method, N-acetylglyphosate less than 200 Nm can be detected.

(Example 4: Detection of natural or low activity of GAT enzyme) The natural or low activity of the GAT enzyme is typically K for about 1 / min of Kcat and 1.5-10 Mm of glyphosate.MHave. K about acetyl-CoAMIs typically less than 25 μM.

Bacterial cultures were grown in nutrient-rich medium in deep 96-well plates, and 0.5 ml quiescent cells were collected by centrifugation and washed with 5 mM morpholine acetate (pH 8). , And suspended in 0.1 ml reaction mixture (in 5 mM morpholine acetate (pH 8)) containing 200 μM ammonium acetyl CoA, 5 mM ammonium glyphosate, and 5 μg / ml PMBS (Sigma). This PMBS can permeable cell membranes and transfer substrates and products from cells to buffer without releasing all of the cellular contents. The reaction was carried out at 25-37 ° C for 1-48 hours. The reaction was rested with equal volume of 100% ethanol and the entire mixture was filtered on a 0.45 μm MAHV Multiscreen filter plate (Millipore). Samples were analyzed using a mass spectrometer as described above and compared to standard synthetic N-acetylglyphosate.

(Example 5: Detection of highly active GAT enzyme) Highly active GAT enzymes typically include kcat up to 400 / min and K below 0.1 mM glyphosate.MHave.

The gene encoding the GAT enzyme was cloned into an E. coli expression vector (eg, pQE80 (Qiagen)) and introduced into the E. coli strain (eg, XL1 Blue (Stratagene)). Cultures were grown to late logarithmic growth in 150 μl nutrient-rich medium (eg, LB with 50 μg / ml carbenicllin) in a shallow U-bottomed 96-well polystyrene plate, and 1 mM IPTG. Diluted 1: 9 with fresh medium containing (USB). After 4-8 hours of induction, cells were collected and 5 mM morpholine acetate pH It was washed with 6.8 and resuspended in the same volume of morpholine buffer. The reaction was performed with washed cells up to 10 μl. At higher activity levels, the cells were first diluted 1: 200 and 5 μl was added to 100 μl of the reaction mixture. To measure GAT activity, the same reaction mixture described for low activity can be used. However, to detect the highly active GAT enzyme, the concentration of glyphosate was lowered to 0.15-0.5 mM, the pH was lowered to 6.8, and the reaction was run at 37 ° C for 1 hour. Reaction work-up and MS detection were as described herein.

(Example 6: Purification of GAT enzyme) Enzyme purification was achieved by affinity chromatography on cell lysates on CoA agarose and gel filtration on Superdex-75. Obtained up to 10 mg of purified GAT enzyme as follows: 100 ml of a culture of E. coli carrying the GAT polynucleotide on a pQE80 vector, grown overnight in LB containing 50 μg / ml carbenicillin. It was used to inoculate 1 L of LB supplemented with 50 μg / ml carbenicillin. IPTG was then added to 1 mM for 1 hour, and the culture was grown for an additional 6 hours. Cells were collected by centrifugation. Dissolution, 25 mM HEPES (pH 7.2), 100 mM KCl, 10% It was brought about by suspending the cells in methanol (referred to as HKM), 0.1 mM EDTA, 1 mM DTT, a prosthesis inhibitor cocktail fed by Sigma-Aldrich and 1 mg / ml chicken egg lysozyme. The cells were then sonicated for a period of time at room temperature for 30 minutes. The granular material was removed by centrifugation and the lysate was passed through a layer of coenzyme A agarose. The column was washed with HKM several times the layer volume and GAT was eluted with HKM 1.5 times the layer volume containing 1 mM acetylcoenzyme A. GAT in the eluate was concentrated by its retention across the Centricon YM 50 ultrafiltration membrane. Further purification, its protein, Superdex via a series of 0.6 ml injections Obtained by passing through 75 columns. The peak of the GAT active eluate was eluted at a volume corresponding to the molecular weight of 17 kD. This method resulted in a homogeneous purification of more than 85% recovery of the GAT enzyme. Similar procedures were used to simultaneously obtain up to 96 shuffled variants in amounts of 0.1-0.4 mg. The volume of the induced culture was reduced to 1-10 ml, coenzyme A-agarose affinity chromatography was performed on a 0.15 ml column packed in MAHV filter plates (Millipore), and Superdex 75 chromatography was omitted.

(Example 7: KcatAnd KMStandard protocol for decision) K for purified protein glyphosatecatAnd KMWas determined using a continuous spectroscopic assay, where the hydrolysis of the sulfoester bond of AcCoA was monitored at 235 nm. The reaction was performed in wells of a 96-well assay plate at ambient temperature (about 23 ° C) with the following components present in a final volume of 0.3 ml: 20 mM HEPES (pH 6.8), 10%. Ethylene glycol, 0.2 mM acetylcoenzyme A, and various concentrations of ammonium gluphosate. In a kinetic comparison of the two GAT enzymes, both enzymes should be assayed under the same conditions (eg, both at 23 ° C). Kcat, VmaxAnd calculated from the enzyme concentration determined by the Bradford assay. KMWas calculated from the initial reaction rate obtained from the glyphosate concentration varying in the range of 0.125 to 10 mM using the Michaelis-Menten formula Rheinweberberg transformation. Kcat/ KM, KcatThe value determined for, KMWas determined by dividing by the determined value.

The methodology was used to determine the velocity parameters for many of the GAT polypeptides exemplified herein. For example, K for the GAT polypeptide corresponding to SEQ ID NO: 445.cat, KMAnd Kcat/ KMWas determined to be 322 / min, 0.5 mM, and 660 / mM / min, respectively, using the above assay conditions. K for the GAT polypeptide corresponding to SEQ ID NO: 457cat, KMAnd Kcat/ KMWas determined to be 118 / min, 0.1 mM, and 1184 / mM / min, respectively, using the above assay conditions. K for the GAT polypeptide corresponding to SEQ ID NO: 300cat, KMAnd Kcat/ KMWas determined to be 296 / min, 0.65 mM, and 456 / mM / min, respectively, using the above assay conditions. One of ordinary skill in the art can use these numbers to confirm that the GAT activity assay produces velocity parameters for GAT that are suitable for comparison with the values given herein. For example, the conditions used to compare GAT activity are SEQ ID NOs: 300, 445, and when the conditions are used to compare a test GAT with a GAT polypeptide exemplified herein. For 457, it should yield the same rate constants (within normal experimental variances) as these values reported herein. Velocity parameters for many GAT polypeptide variants were determined according to this methodology and provided in Tables 3, 4, and 5.

(Table 3. GAT polypeptide kcatvalue)

<tables num="3"><img file="JP2010142234A_D0003.tif" /></tables><img file="JP2010142234A_D0004.tif" /><img file="JP2010142234A_D0005.tif" /><img file="JP2010142234A_D0006.tif" /><img file="JP2010142234A_D0007.tif" /> (Table 4. GAT Polypeptide (Glyphosate) KMvalue)

<tables num="4"><img file="JP2010142234A_D0008.tif" /></tables><img file="JP2010142234A_D0009.tif" /><img file="JP2010142234A_D0010.tif" /><img file="JP2010142234A_D0011.tif" /><img file="JP2010142234A_D0012.tif" /> (Table 5. GAT polypeptide kcat/ KMvalue)

<tables num="5"><img file="JP2010142234A_D0013.tif" /></tables><img file="JP2010142234A_D0014.tif" /><img file="JP2010142234A_D0015.tif" /><img file="JP2010142234A_D0016.tif" /><img file="JP2010142234A_D0017.tif" /> K about AcCoAMWas measured using mass spectrometry with repeated sampling over the reaction. Acetyl coenzyme A and glyphosate (ammonium salt) were placed in the wells of a mass spectrometer sample plate as a 50-fold stock solution. The reaction was initiated by the addition of an enzyme appropriately diluted in volatile buffer (pH 6.8 or 7.7) such as morpholine acetate or ammonium carbonate. The sample was repeatedly injected into the instrument and the initial velocity was calculated from the retention time and peak area plots. KMWas calculated in the same manner as for glyphosate.

(Example 8: Selection of transformed E. COLI) The evolved gat gene (chimeric with the native B. licheniformis ribosome binding site (AACTGAAGGAGGAATCTC; SEQ ID NO: 515) directly bound to the 5'end of the GAT coding sequence) is expressed in the expression vector pQE80 between the EcoRI and HindIII sites. It was cloned into (Qiagen) to give the plasmid pMAXY2190 (Fig. 11). It eliminated the His-tag domain from this plasmid and carried the B-lactamase gene, which conferred resistance to the antibiotics ampicillin and carbenicillin. pMAXY2190 was electroporated (Bio Rad Gene Pulser) into XL1 Blue (Stratagene) E. coli cells. The cells were suspended in SOC-rich medium and allowed to recover for 1 hour. The cells are then gradually pelleted and M9 minimal medium lacking aromatic amino acids (12.8 g / L Na).2HPO4 7H2O, 3.0g / L KH2PO4, 0.5g / L NaCl, 1.0g / L NH4Cl, 0.4% glucose, 2 mM DDL4, 0.1 mM CaCl2, 10 ml / L thiamine, 10 mg / L proline, 30 mg / L carbenicillin) and resuspended in 20 ml of the same M9 medium. After overnight growth at 250 rpm at 37 ° C, equivolume cells were plated on either M9 medium or M9 plus 1 mM glyphosate. The pQE80 vector without the gat gene was similarly introduced into E. coli cells and plated into a single colony for comparison. The results are summarized in Table 6. And this result clearly shows that GAT activity allows selection and proliferation of transformed E. coli with less than 1% background. Note that IPTG induction is not essential for sufficient GAT activity to allow the proliferation of transformed cells. Transformation was confirmed by reisolation of pMAXY2190 derived from E. coli cells grown in the presence of glyphosate.

<tables num="6"><img file="JP2010142234A_D0018.tif" /></tables> (Example 9: Selection of transformed plant cells) Agrobacterium-mediated transformation of plant cells occurs with low efficacy. Selectable markers are needed to allow the growth of transformed cells while inhibiting the growth of non-transformed cells. Antibiotic markers for kanamycin and hygromycin, as well as herbicides that modify the gene bar, which detoxifies the herbicide compound phosphinothricin, are examples of selectable markers used in plants. (Methods in Molecular Biology, 1995, 49: 9 ~ 18). Here, we demonstrate that GAT activity serves as an effective selectable marker for plant transformation. The evolved gat gene (0_5B8) was cloned between the plant promoter (enhanced strawberry vein stripe virus) and the ubiquinone terminator, and Agrobacterium tumefaciens, as shown in FIG. It was introduced into the T-DNA region of a two-component vector pMAXY3793 suitable for plant cell transformation via EHA105. Screenable GUS markers were present in T-DNA, allowing confirmation of transformation. Glyphosate was used as the only drug of choice to produce transgenic tobacco shoots.

Axillary buds of Nicotiana tabacum L. Xanthi, 16 hours of light (35-42 μE insteins m)-2s-1Subculture in half-strength MS medium containing sucrose (1.5%) and gelrite (0.3%) every 2-3 weeks at 24 ° C. under white fluorescent light. did. After 2-3 weeks of subculture, young leaves were excised from the plant and cut into 3 x 3 mm pieces. A.tumefaciens EHA105 was inoculated into LB medium and grown overnight to a density of A600 = 1.0. Pellet cells at 4,000 rpm for 5 minutes, Murashige and containing 2 mg / L N6-benzyladenine (BA) Resuspended in 3 volumes of liquid co-culture medium containing Skoog (MS) medium (pH 5.2), 1% glucose and 400 uM acetysyringone. The leaf pieces were then completely immersed in 20 ml of A. tumefaciens in a 100 x 25 mm Petri dish for 30 minutes, blotted with autoclaved filter paper, and then placed in solid co-culture medium (0.3% gellite). , And incubated as described above. After 3 days of co-culture, 20-30 fragments were taken from MS solid medium (pH 5.7) containing 2 mg / L BA, 3% sucrose, 0.3% gellite, 0-200 uM glyphosate, and 400 ug. Transferred to basal shoot-induction (BSI) medium containing / ml Timentin.

After 3 weeks, shoots were clearly present on shoots placed in glyphosate-free medium, with or without the gat gene. T-DNA transfer from both constructs was confirmed by GUS histochemical staining of leaves from regenerated shoots. Glyphosate concentrations greater than 20uM completely inhibited any shoot formation from grafts lacking the gat gene. Grafts infected with A. tumefaciens with gat constructs regenerated shoots at glyphosate concentrations up to 200 uM (highest level tested). Transformation was confirmed by GUS histochemical staining and by PCR fragment amplification of the gat gene with primers annealing to the promoter and 3'region. The results are summarized in Table 7.

<tables num="7"><img file="JP2010142234A_D0019.tif" /></tables> (Example 10: Glyphosate selection of transformed yeast cells) Selectable markers for yeast transformation are usually auxotrophic genes that allow the growth of transformed cells in media lacking certain amino acids or nucleotides. Since Saccharomyces cerevisiae is sensitive to glyphosate, GAT can also be used as a selectable marker. To demonstrate this, the evolved gat gene (0_6D10) was cloned from the T-DNA vector pMAXY3793 (as shown in Example 9) as a PstI-ClaI fragment containing the entire coding region and shown in FIG. Ligate to PstI-ClaI digestion p424TEF (Gene, 1995, 156: 119 ~ 122) as shown in. This plasmid contains the origin of replication of E. coli, a gene conferring carbenicillin resistance, and TRP1 (tryptophan auxotrophy selectable marker for yeast transformation).

The gat-containing construct is transformed into E. coli XL1 Blue (Statagene) and plated on LB carbenicillin (50 ug / ml) agar medium. Plasmid DNA is prepared and used to transform the yeast strain YPH499 (Stratagene) with a transformation kit (Bio101). Equal amounts of transformed cells are plated on CSM-YNB-glucose medium (Bio101), which lacks all aromatic amino acids (tryptophan, tyrosine, and phenylalanine) and is glyphosate-supplemented. For comparison, p424TEF lacking the gat gene is also introduced into YPH499 and plated as above. This result demonstrates that GAT activity functions as an effective selectable marker. The presence of the gat-containing vector in glyphosate-selected colonies can be confirmed by plasmid reisolation and restriction digestion analysis.

Although the invention described above has been described in some detail for the purpose of clarity and understanding, those skilled in the art can make various changes in form or detail without departing from the true scope of the invention. For those of you who read this disclosure, it is clear. For example, all the techniques, methods, compositions, devices and systems described above can be used in various combinations. The present invention is the product of all methods and reagents described herein, as well as all polynucleotides, polypeptides, cells, organisms, plants, grains (these are the products of these novel methods and reagents). ) Etc. are intended to be included.

All publications, patents, patent applications, or other documents cited in this application are incorporated herein by reference in their respective publications, patents, patent applications, or other documents for all purposes. Incorporated in its entirety herein as a reference for all purposes to the same extent as indicated individually as incorporated.

108 members in 23 offices

This recordMember, by publication datePriority claim13 more offices are in the list below

Priority claims5

Priority claims
Document	Office	Kind	Date
24438500	United States of America	P	2000-10-30
24438500	United States of America	P	2000-10-30
60244385	United States of America	–	2000-10-30
2000244385	–	–	–
US20000244385P	–	–	–

Members108

Family members, oldest first
Document	Office	Kind
CA2425956A1	Canada	A1	2002-05-10
WO0236782A2	World Intellectual Property Organization (WIPO)	A2	2002-05-10
AU2018102A	Australia	A	2002-05-15
US2003083480A1	United States of America	A1	2003-05-01
CZ20031120A3	Czechia	A3	2003-11-12
WO03092360A2	World Intellectual Property Organization (WIPO)	A2	2003-11-13
AU2003234328A1	Australia	A1	2003-11-17
AU2003234328A8	Australia	A8	2003-11-17
WO0236782A3	World Intellectual Property Organization (WIPO)	A3	2004-01-08
EP1399566A2	European Patent Office (EPO)	A2	2004-03-24
US2004082770A1	United States of America	A1	2004-04-29
AR035595A1	Argentina	A1	2004-06-16
BG107758A	Bulgaria	A	2004-07-30
CN1531594A	China	A	2004-09-22
MXPA03003810A	Mexico	A	2004-10-15
JP2004534505A	Japan	A	2004-11-18
SK5222003A3	Slovakia	A3	2004-12-01
PL366144A1	Poland	A1	2005-01-24
AU2004260931A1	Australia	A1	2005-02-10
CA2521284A1	Canada	A1	2005-02-10
CA2662092A1	Canada	A1	2005-02-10
WO2005012515A2	World Intellectual Property Organization (WIPO)	A2	2005-02-10
AR039501A1	Argentina	A1	2005-02-23
WO03092360A3	World Intellectual Property Organization (WIPO)	A3	2005-03-17
BR0115046A	Brazil	A	2005-04-12
WO2005012515A8	World Intellectual Property Organization (WIPO)	A8	2005-05-12
ZA200303138B	South Africa	B	2005-06-29
AR044141A1	Argentina	A1	2005-08-24
NZ526148A	New Zealand	A	2005-09-30
WO2005012515A3	World Intellectual Property Organization (WIPO)	A3	2005-11-17
EP1620557A2	European Patent Office (EPO)	A2	2006-02-01
HRP20050930A2	Croatia	A2	2006-03-31
BRPI0409816A	Brazil	A	2006-05-02
MXPA05011585A	Mexico	A	2006-05-25
US2006191033A1	United States of America	A1	2006-08-24
US2006200874A1	United States of America	A1	2006-09-07
US2006218663A1	United States of America	A1	2006-09-28
CN1863914A	China	A	2006-11-15
RS32703A	Serbia	A	2006-12-15
US2007004912A1	United States of America	A1	2007-01-04
JP2007500514A	Japan	A	2007-01-18
ZA200509602B	South Africa	B	2007-04-25
HU0700153A2	Hungary	A2	2007-08-28
HUP0700153A2	Hungary	A2	2007-08-28
AU2007205733A1	Australia	A1	2007-08-30
AU2007224390A1	Australia	A1	2007-11-01
AU2002220181B2	Australia	B2	2007-12-20
RS20050804A	Serbia	A	2007-12-31
US2008119639A1	United States of America	A1	2008-05-22
JP2008206519A	Japan	A	2008-09-11
AU2007205733B2	Australia	B2	2008-11-20
US7462481B2	United States of America	B2	2008-12-09
HRP20030439A2	Croatia	A2	2008-12-31
US2009011938A1	United States of America	A1	2009-01-08
AR064756A2	Argentina	A2	2009-04-22
US7527955B2	United States of America	B2	2009-05-05
US7531339B2	United States of America	B2	2009-05-12
AU2009201716A1	Australia	A1	2009-05-21
UA86918C2	Ukraine	C2	2009-06-10
CL2009000280A1	Chile	A1	2009-09-25
NZ543826A	New Zealand	A	2009-09-25
SG155063A1	Singapore	A1	2009-09-30
US2009260104A1	United States of America	A1	2009-10-15
US2009282586A1	United States of America	A1	2009-11-12
AU2004260931B2	Australia	B2	2009-11-19
US2009298714A1	United States of America	A1	2009-12-03
US2009325804A1	United States of America	A1	2009-12-31
AR069485A2	Argentina	A2	2010-01-27
CL2009001733A1	Chile	A1	2010-02-05
US7666643B2	United States of America	B2	2010-02-23
CN101665795A	China	A	2010-03-10
AR070289A2	Argentina	A2	2010-03-25
CN101684458A	China	A	2010-03-31
US7709702B2	United States of America	B2	2010-05-04
US7714188B2	United States of America	B2	2010-05-11
JP2010142234AThis record	Japan	A	2010-07-01
US2010184204A1	United States of America	A1	2010-07-22
US2010199371A1	United States of America	A1	2010-08-05
NZ575248A	New Zealand	A	2010-09-30
JP2010220617A	Japan	A	2010-10-07
JP2010227104A	Japan	A	2010-10-14
CN1863914B	China	B	2011-03-09
AU2007224390B2	Australia	B2	2011-03-17
EP2322629A2	European Patent Office (EPO)	A2	2011-05-18
CN1531594B	China	B	2011-05-25
UA94688C2	Ukraine	C2	2011-05-25
US7998703B2	United States of America	B2	2011-08-16
US7999152B2	United States of America	B2	2011-08-16
US8008547B2	United States of America	B2	2011-08-30
US8021857B2	United States of America	B2	2011-09-20
IL155599A	Israel	A	2011-09-27
CN102212534A	China	A	2011-10-12
US8044261B2	United States of America	B2	2011-10-25
EP2322629A3	European Patent Office (EPO)	A3	2011-11-02
US8088972B2	United States of America	B2	2012-01-03
AU2004260931B9	Australia	B9	2012-01-19
AU2009201716B2	Australia	B2	2012-05-17
US2012122686A1	United States of America	A1	2012-05-17
CA2662092C	Canada	C	2012-07-17
IL191899A	Israel	A	2012-08-30

Showing 100 of 108

2 legal events, as the office reported them to INPADOC

Over the term

Point at a mark for the event

Events

Legal events, newest first
	Event	Code
2011-06-07	Application deemed to be withdrawn because no request for examination was validly filedWithdrawnA300	A300
2011-05-16	Dismissal of procedure [no reply to invitation to correct request for examination]A072	A072