Glyphosate tolerant wheat plant 33391 and compositions and methods for detection thereof
20 claims: 15 independent, 5 dependent
- 1Sposób wytwarzania tolerancyjnej na glifosat rośliny pszenicy, znamienny tym, że zawiera etapy w których:(1) uzyskuje się konstrukt DNA zawierający pierwszą i drugą kasetę ekspresji, przy czym wspomniana pierwsza kaseta ekspresji w operacyjnym połączeniu zawiera: (i) promotor aktyny 1 ryżu;(ii) intron aktyny 1 ryżu;(iii) cząsteczkę DNA kodującą tranzytowy peptyd chloroplastu;(iv) cząsteczkę DNA kodującą EPSPS z tolerancją glifosatu;i (v) cząsteczkę DNA terminatora transkrypcji;a wspomniana druga kaseta ekspresji zawiera w połączeniu operacyjnym (a) starter CaMV 35S;(b) intron Hsp70;(c) cząsteczkę DNA kodującą tranzytowy peptyd chloroplastu;(d) cząsteczkę DNA kodującą EPSPS z tolerancją glifosatu;i (e) cząsteczkę DNA terminatora transkrypcji;i (2) transformuje się komórki pszenicy wspomnianym konstruktem DNA;i (3) regeneruje się wspomnianą komórkę pszenicy do rośliny pszenicy;i (4) traktuje się wspomnianą roślinę pszenicy skuteczną dawką glifosatu;i (5) wybiera się płodne rośliny pszenicy, które wegetatywnie i reprodukcyjnie tolerują glifosat i zawierają cząsteczkę DNA zawierającą nukleotydy 245-270 SEQ ID nr 5 albo nukleotydy 87-113 SEQ ID nr 6.
- 2Płodna tolerująca glifosat roślina pszenicy wytworzona sposobem, jak określono w zastrz. 1.
- 3Nasiono pszenicy wytworzone sposobem, jak określono w zastrz. 1.
- 4Para cząsteczek DNA zawierająca:pierwszą cząsteczkę DNA i drugą cząsteczkę DNA, przy czym pierwsza cząsteczka DNA zawiera co najmniej 8 sąsiadujących ze sobą nukleotydów od nukleotydu 1 do 257 SEQ ID nr: 5 lub jej pełnego komplementu, a druga cząsteczka DNA zawiera co najmniej 8 sąsiadujących ze sobą nukleotydów od nukleotydu 258 do 399 z SEQ ID nr: 5 lub jej pełnego komplementu, i ta para cząsteczek DNA kiedy jest stosowana w metodzie amplifikacji DNA wytwarza amplikon obejmujący nukleotydy 245-270 o sekwencji SEQ ID nr: 5
- 5Para cząsteczek DNA zawierająca:pierwszą cząsteczkę DNA i drugą cząsteczkę DNA, przy czym pierwsza cząsteczka DNA zawiera co najmniej 8 sąsiadujących ze sobą nukleotydów od nukleotydu 1 do 100 z SEQ ID nr: 6 pszenicy lub jej pełnego komplementu, a druga cząsteczka DNA zawiera co najmniej 8 sąsiadujących ze sobą nukleotydów od nukleotydu 101 do 431 z SEQ ID nr: 6 lub jej pełnego komplementu, i ta para cząsteczek DNA kiedy jest stosowana w metodzie amplifikacji DNA wytwarza amplikon obejmujący nukleotydy 87-113 sekwencji SEQ ID nr: 6.
- 6Sposób wykrywania obecności diagnostycznej cząsteczki DNA dla tolerującej glifosat rośliny pszenicy zawierającej nukleotydy 245-270 SEQ ID nr 5 albo nukleotydy 87-113 SEQ ID nr 6 lub jej potomstwa, znamienny tym, że obejmuje etapy w których:(a) ekstrahuje się próbkę DNA ze wspomnianej rośliny pszenicy lub jej nasion potomnych i roślin potomnych lub ich części;i (b) dostarcza się cząsteczki DNA startera jak zdefiniowano w zastrz. 4 lub zastrz. 5;i (c) zapewnia się warunki amplifikacji DNA;i (d) przeprowadza się wspomnianą reakcję amplifikacji DNA, wytwarzając w ten sposób cząsteczkę DNA amplikonu;i (e) wykrywa się cząsteczkę DNA amplikonu.
- 7Para cząsteczek DNA według zastrz. 4, w której wspomniane cząsteczki DNA zawierają SEQ ID nr:7 i SEQ ID nr: 8.
- 8Sposób wykrywania sekwencji zawierającej nukleotydy 245-270 SEQ ID nr 5 albo nukleotydy 87-113 SEQ ID nr 6 w próbce DNA, znamienny tym, że sposób zawiera etapy, w których:PL 214 848 B1 (a) ekstrahuje się próbkę DNA z rośliny pszenicy;(b) kontaktuje się próbki DNA z cząsteczką DNA, zawierającą nukleotydy 245-270 z sekwencji SEQ ID nr: 5;lub nukleotydy 87-113 z SEQ ID nr: 6, i (c) poddaje się próbkę i sondę ostrym warunkom hybrydyzacji, i (d) wykrywa się hybrydyzację sondy z tym DNA.
- 9Sposób wytwarzania rośliny pszenicy z tolerancją glifosatu, znamienny tym, że:zawiera etapy w których (a) krzyżuje się potomstwo pszenicy tolerującej glifosat zawierającej sekwencję zawierającą nukleotydy 245-270 SEQ ID nr 5 albo nukleotydy 87-113 SEQ ID nr 6 z roślinami pszenicy nie tolerującymi glifosatu;i (b) wytwarza się potomstwo roślin pszenicy tej krzyżówki;(c) ekstrahuje się próbki DNA z potomstwa roślin pszenicy;(d) kontaktuje się próbki DNA z parą starterów jak określono w zastrz. 4 lub zastrz. 5;(e) przeprowadza się reakcję amplifikacji DNA;i (f) selekcjonuje się potomstwo roślin pszenicy, które produkuje amplikon obejmujący nukleotydy 245-270 z SEQ ID nr: 5 lub nukleotydy 87-113 z SEQ ID nr: 6.
- 10Zestaw do wykrywania DNA, znamienny tym, że zawiera kontrolę pozytywną dla sekwencji zawierającej nukleotydy 245-270 SEQ ID nr 5 albo nukleotydy 87-113 SEQ ID nr 6; i albo (a) wspomnianą parę starterów jak zdefiniowano w zastrz. 4 lub 5; (b) sondę zawierającą fragmenty sekwencji SEQ ID nr:5 zawierającą nukleotydy 245-270 o sekwencji SEQ ID nr: 5;albo (c) sondę zawierającą fragmenty sekwencji SEQ ID nr: 6 zawierającą nukleotydy 87-113 o sekwencji SEQ ID nr: 6.
- 11Nasiono transgenicznej pszenicy zwierające sekwencję zawierającą nukleotydy 245-270 SEQ ID nr 5 albo nukleotydy 87-113 SEQ ID nr 6, przy czym genom wspomnianego nasiona obejmuje insert DNA zawierający dwie kopie cząsteczki DNA kodującego EPSPS z tolerancją glifosatu, gdzie wspomniany insert posiada połączenie 5' z genomowym DNA pszenicy obejmujące SEQ ID nr:5 oraz posiada połączenie 3' z genomowym DNA pszenicy obejmujące SEQ ID nr: 6.
- 12Roślina pszenicy wytworzona przez hodowlę nasion jak zdefiniowano w zastrz. 11.
- 13Sposób wytwarzania rośliny pszenicy tolerującej stosowanie glifosatu, znamienny tym, że zawiera etapy w których:(a) krzyżuje się płciowo pierwszą roślinę pszenicy zawierającą sekwencję zawierającą nukleotydy 245-270 SEQ ID nr 5 albo nukleotydy 87-113 SEQ ID nr 6 z drugą rośliną pszenicy z brakiem tolerancji na glifosat, wytwarzając przez to mnogość roślin pierwszego pokolenia;(b) wybiera się roślinę pierwszego pokolenia, (c) doprowadza się do samozapylenia wspomnianą roślinę pierwszego pokolenia, wytwarzając w ten sposób mnogość roślin drugiego pokolenia;(d) nanosi się herbicyd glifosat na wspomnianą roślinę drugiego pokolenia;(e) wybiera się ze wspomnianych roślin drugiego pokolenia roślinę tolerującą glifosat.
- 14Sposób selektywnego kontrolowania chwastów na polu zawierającym uprawę pszenicy, znamienny tym, że zawiera etapy, w których:(a) sadzi się nasiona jak określono w zastrz. 11, i (b) nanosi się na uprawę pszenicy i chwasty na polu wystarczającą ilość herbicydu glifosatu dla kontrolowania chwastów bez znacznego uszkadzania uprawy pszenicy.
- 15Cząsteczka DNA obejmująca SEQ ID nr:5 lub jej fragment zawierający nukleotydy 245-270 z SEQ ID nr: 5.
- 16Cząsteczka DNA obejmująca SEQ ID nr:6 lub jej fragment zawierający nukleotydy 87-113 z SEQ ID nr: 6.
- 17Roślina pszenicy tolerująca glifosat, której genom zawiera insert DNA zawierający dwie kasety ekspresyjne kodujące oporną na glifosat syntazę 5-enolopirogroniano-szikimowo-3-fosforanową (EPSPS) z Agrobacterium tumefaciens (ARGl) sp. Szczep CP4 i sekwencje połączenia spinające miejsce insertu zawierające nukleotydy 245-270 o SEQ ID nr:5 i nukleotydy 87-113 o sekwencji SEQ ID nr: 6.
- 18Roślina pszenicy zdolna do wytwarzania diagnostycznego amplikonu obejmującego SEQ ID nr:5 lub SEQ ID nr: 6. PL 214 848 B1
- 19Nasiono zdolne do wytwarzania diagnostycznego amplikonu obejmującego SEQ ID nr:5 lub SEQ ID nr: 6.
- 20Komórka zdolna do wytwarzania diagnostycznego amplikonu obejmującego SEQ ID nr:5 lub SEQ ID nr: 6.
Independent claims20
153 paragraphs in 7 sections, as filed
The invention relates to a method of producing a glyphosate-tolerant wheat plant and a fertile glyphosate-tolerant wheat plant and wheat seed produced by the method. The present invention also relates to a pair of DNA molecules, a method for detecting the presence of a diagnostic DNA molecule for a glyphosate-tolerant wheat plant, a method for detecting a sequence containing certain nucleotides, and a method for producing a glyphosate-tolerant wheat plant, a DNA detection kit, transgenic wheat seed, and culture-produced wheat plant. the above seeds. In addition, the invention relates to a method of producing a glyphosate tolerant wheat plant, a method of selectively controlling weeds in a field containing a wheat crop, a DNA molecule, and a glyphosate tolerant wheat plant, and a plant, seed, cell capable of producing a diagnostic amplicon.
Wheat is an important crop and the staple food source in many parts of the world. Biotechnological methods are used to improve the agronomic and qualitative characteristics of wheat. One such agronomic characteristic is herbicide tolerance, in particular tolerance to the glyphosate herbicide. This trait in wheat is conferred by the expression of the transgene in wheat plants (Zhou et al., Plant Cell rep. 15: 159-163, 1995). Expression of foreign genes in plants is known to be influenced by their chromosomal position, sometimes due to the structure of chromatin (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g. enhancers) close to the integration site (Weising et al., Ann. Rev. Genet 22: 421-477,188). For this reason, it is often necessary to study a large number of cases in order to identify a case characterized by the optimal expression of the desired gene inserted. For example, it has been observed in plants and other organisms that there can be wide variation in the expression levels of the introduced gene between events. There may also be differences in the spatial and temporal expression pattern, for example, differences in the relative expression of the transgene in different plant tissues, which may not coincide with patterns expected from transcriptional regulatory elements present in the introduced gene construct. For this reason, it is common to generate hundreds to thousands of different cases and study these cases to detect that one case having the desired levels of transgene and pattern expression for commercial purposes. An event having a desired transgene expression level or pattern is useful for introgressing that transgene into a different genetic background by sexual outcrossing using conventional breeding methods. The progeny of such crosses support the transgene expression characteristics of the original transformant. This strategy is used to ensure some expression of the gene in many variants that are better adapted to local growth conditions.
It would be advantageous to be able to detect the presence of individual cases in order to determine whether the progeny of a sexual cross contains the transgene of interest. In addition, a case-by-case detection method would assist in complying with the pre-approval and labeling regulations for food derived for example from recombinant crops. It is possible to detect the presence of the transgene by any well known method of nucleic acid detection such as polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements such as promoters, terminators, marker genes, etc. As a result, these methods may not be useful for distinguishing between different cases, especially those produced by using the same DNA construct, unless the sequence of the chromosomal DNA adjacent to the inserted ("flanking DNA") is known. Case-specific PCR testing is discussed, for example, by Windels et al. (Med. Fac. Landbouww, Univ. Gent 64 / 5b: 459-462,1999), which identified a glyphosate-tolerant soybean 40-3-2 case by PCR using a set of primers linking the bond between the insert and the flanking DNA, especially a primer that contained the sequence from this insert and a second primer which contained the flanking DNA sequence.
The present invention relates to an improved glyphosate herbicide tolerant wheat 33391 (Triticum aestivum) plant and plant DNA 33391 expression construct of the wheat plant, and detection of the transgenic / genomic insert region in wheat 33391 and its progeny.
The invention relates to a method of producing a glyphosate-tolerant wheat plant comprising the steps of:
(1) obtaining a DNA construct comprising first and second expression cassettes, said first expression cassette in operable linkage comprising:
(i) rice actin 1 promoter;
(ii) a rice actin 1 intron;
(iii) a DNA molecule encoding a chloroplast transit peptide;
(iv) a DNA molecule encoding glyphosate tolerant EPSPS; and (v) a DNA transcription terminator molecule; and said second expression cassette comprises in the operative link (a) a CaMV 35S primer;
(b) an Hsp70 intron;
(c) a DNA molecule encoding a chloroplast transit peptide;
(d) a DNA molecule encoding glyphosate tolerant EPSPS; and (e) a transcription terminator DNA molecule; and (2) transforming wheat cells with said DNA construct; and (3) regenerating said wheat cell into a wheat plant; and (4) treating said wheat plant with an effective amount of glyphosate; and (5) selecting fertile wheat plants that vegetatively and reproductively tolerate glyphosate and contain a DNA molecule comprising nucleotides 245-270 of SEQ ID No. 5 or nucleotides 87-113 of SEQ ID No. 6.
The invention also relates to a glyphosate-tolerant fertile wheat plant produced by the method as defined above.
The invention relates to wheat seed produced by the method as defined above.
The invention relates to a pair of DNA molecules comprising: a first DNA molecule and a second DNA molecule, the first DNA molecule having 8 contiguous nucleotides from nucleotide 1 to 257 of SEQ ID NO: 5 or a complete complement thereof, and the second DNA molecule contains at least 8 contiguous nucleotides from nucleotide 258 to 399 of SEQ ID NO: 5 or the full complement thereof, and said pair of DNA molecules when used in a DNA amplification method produces an amplicon comprising nucleotides 245-270 of SEQ ID NO: 5.
The invention relates to a pair of DNA molecules comprising: a first DNA molecule and a second DNA molecule, the first DNA molecule having at least 8 contiguous nucleotides from nucleotides 1 to 100 of SEQ ID NO: 6 of wheat or its full complement, and the second DNA molecule comprises at least 8 contiguous nucleotides from nucleotide 101 to 431 of SEQ ID NO: 6 or the full complement thereof, and said pair of DNA molecules when used in a DNA amplification method produces an amplicon comprising nucleotides 87-113 of SEQ ID NO: 6.
The invention also relates to a method for detecting the presence of a diagnostic DNA molecule for a glyphosate-tolerant wheat plant comprising nucleotides 245-270 of SEQ ID NO: 5 or nucleotides 87-113 of SEQ ID NO: 6 or its progeny comprising the steps of:
(a) extracting a DNA sample from said wheat plant or its progeny and daughter plants or parts thereof; and (b) providing DNA primer molecules as defined above; and (c) providing DNA amplification conditions; and (d) performing said DNA amplification reaction, thereby producing an amplicon DNA molecule; and (e) detecting the amplicon DNA molecule.
Preferably said DNA molecules comprise SEQ ID NO: 7 and SEQ ID NO: 8.
The invention relates to a method for detecting a sequence comprising nucleotides 245-270 of SEQ ID NO: 5 or nucleotides 87-113 of SEQ ID NO: 6 in a DNA sample, characterized in that the method comprises the steps of:
(a) extracting a DNA sample from a wheat plant;
(b) contacting the DNA sample with a DNA molecule comprising nucleotides 245-270 of SEQ ID NO: 5; or nucleotides 87-113 of SEQ ID NO: 6 and (c) subjecting the sample and probe to stringent hybridization conditions, and (d) detecting hybridization of the probe with said DNA.
The present invention relates to a method for producing a glyphosate tolerant wheat plant comprising:
(A) crossing glyphosate-tolerant wheat progeny containing a sequence comprising nucleotides 245-270 of SEQ ID No. 5 or nucleotides 87-113 of SEQ ID No. 6 with glyphosate-intolerant wheat plants; and (b) progeny of the wheat plants of the cross are produced;
(c) extracting DNA samples from the progeny of wheat plants;
(d) contacting the DNA samples with the primer pair indicated above;
(e) performing a DNA amplification reaction; and (f) selecting progeny of wheat plants that produce an amplicon comprising nucleotides 245-270 of SEQ ID NO: 5 or nucleotides 87-113 of SEQ ID NO: 6.
The invention relates to a DNA detection kit comprising a positive control for a sequence comprising nucleotides 245-270 of SEQ ID No. 5 or nucleotides 87-113 of SEQ ID No. 6; and or (a) said primer pair as defined in claim 1; 3 or 4; (b) a probe comprising fragments of the sequence of SEQ ID NO: 5 comprising nucleotides 245-270 of SEQ ID NO: 5; or (c) a probe comprising fragments of the sequence of SEQ ID NO: 6 comprising nucleotides 87-113 of SEQ ID NO: 6.
The invention relates to a transgenic wheat seed comprising sequences comprising nucleotides 245-270 of SEQ ID No. 5 or nucleotides 87-113 of SEQ ID No. 6, wherein the genome of said seed comprises a DNA insert comprising two copies of a DNA molecule encoding glyphosate tolerant EPSPS, wherein said insert has a 5 'link to wheat genomic DNA comprising SEQ ID NO: 5, and a 3' link to wheat genomic DNA comprising SEQ ID NO: 6.
The invention relates to a wheat plant produced by growing a seed as defined above.
The present invention relates to a process for the production of a glyphosate tolerant wheat plant comprising the steps of:
(a) sexually crossing a first wheat plant comprising a sequence comprising nucleotides 245-270 of SEQ ID No. 5 or nucleotides 87-113 of SEQ ID No. 6 with a second glyphosate intolerant wheat plant thereby producing a plurality of first generation plants;
(b) selecting a plant of the first generation; (c) selfing said plant of the first generation, thereby producing a plurality of plants of the second generation;
(d) applying a glyphosate herbicide to said second generation plant;
(e) selecting a glyphosate tolerant plant from said second generation plants.
The present invention relates to a method of selectively controlling weeds in a field containing wheat, comprising the steps of:
(a) the seeds are planted as defined above; and (b) sufficient glyphosate herbicide is applied to the wheat crop and the weeds in the field to control the weeds without significantly damaging the wheat crop.
The invention relates to a DNA molecule comprising SEQ ID NO: 5 or a fragment thereof comprising nucleotides 245-270 of SEQ ID NO: 5.
The invention relates to a DNA molecule comprising SEQ ID NO: 6 or a fragment thereof comprising nucleotides 87-113 of SEQ ID NO: 6.
The invention relates to a glyphosate-tolerant wheat plant whose genome contains a DNA insert containing an expression cassette encoding glyphosate-resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium tumefaciens (ARGT) sp. CP4 strain and junction sequences spanning the insert site containing nucleotides of the insert 245 -270 of SEQ ID NO: 5 and nucleotides 87113 of SEQ ID NO: 6.
The invention relates to a wheat plant capable of producing a diagnostic amplicon comprising SEQ ID NO: 5 or SEQ ID NO: 6.
The invention relates to a seed capable of producing a diagnostic amplicon comprising SEQ ID NO: 5 or SEQ ID NO: 6.
The invention relates to a cell capable of producing a diagnostic amplicon comprising SEQ ID NO: 5 or SEQ ID NO: 6.
In one aspect, the DNA construct is delivered when it is expressed in the cells of the wheat plant and imparts to the wheat plant improved tolerance to the glyphosphate herbicide. Methods of producing and selecting a wheat plant with glyphosate tolerance were presented,
PL 214 848 B1 containing the construct pMON30139. The pMON30139 DNA construct consists of two transgenic expression cassettes. The first expression cassette is the rice actin 1 promoter (Oryzae sativa) (P-Os.Act1) and the intron (I-Os.Act1) operably linked to the EPSPSc chloroplast transit peptide sequence (TS-At.EPSPS) operably linked to the gene (AGRTU .aroA: CP4) encoding glyphosate resistant 5-enolopyruviloshikimato-3-phosphate synthase (EPSPS) isolated from Agrobacterium tumefaciens (AGRTU) sp. strain CP4, operably linked to the nopaline synthase transcription terminator (T-AGRTU.nos). The second transgene expression cassette is the cauliflower mosaic virus (CaMV) 35S (P-CaMV.35S: en) promoter containing a tandem doubling of the enhancer region operably linked to the Zea mays Hsp70 intron (I-Zm.Hsp70) operably linked to the nucleic acid sequence encoding Arabidopsis thaliana EPSPS chloroplast transit peptide sequence, operatively linked to the gene encoding glyphosate resistant 5-enolopyruviloshikimato-3-phosphate synthase (EPSPS) isolated from Agrobacterium tumefaciens sp. strain CP4, operatively linked to the nopaline synatase transcription terminator. These expression cassettes are in tandem and are flanked by DNA regions that contain Agrobacterium tumefaciens DNA sequences (RB and LB) as components of the process used in Agrobacterium methods to insert the expression cassettes into the wheat genome.
In accordance with another aspect, 33391 wheat seed comprising DNA molecules as deposited with the ATCC under accession number # PTA-2347 is also disclosed. The disclosure thus relates to wheat 33391 seed, 33391 wheat plant, 33391 wheat plant part that contains pollen and an egg, and methods for producing an improved glyphosate tolerant wheat plant by crossing the 33391 wheat plant with itself or another wheat plant.
In another aspect, compositions and methods for detecting the presence of the transgenic / genomic insert region from wheat 33391 plants and seeds are also provided. According to one aspect, DNA molecules are provided that comprise at least one insert region transgene / genomic sequence 33391 wheat selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 6 and their complements, wherein the insertion region sequence is a link between heterologous DNA inserted into the wheat genome and DNA from the wheat genome flanking this insertion site and is diagnostic for this case. Included are DNA sequences that contain a sufficient length of polynucleotides of the transgene insert sequence and sufficient length of polynucleotides of the wheat genomic sequence 33391 of SEQ ID NO: 5, which are useful as primer sequences for the production of the wheat diagnostic product amplicon 33391. Included are DNA sequences that contain a sufficient length of the transgene insert sequence polynucleotides and a sufficient length of the wheat genomic sequence 33391 polynucleotides of SEQ ID NO: 6, which are useful as primer sequences for producing the wheat 33391 diagnostic amplicon product.
According to another aspect, DNA molecules that are diagnostic for wheat 33391 are provided. This aspect is directed to wheat 33391 containing at least one novel DNA molecule. DNA molecules containing nucleic acid primers are provided that provide at least one novel product - wheat amplicon 33391 comprising SEQ ID NO: 7 and SEQ ID NO: 8, or the complements thereof. These DNA amplicons are diagnostic for wheat 33391. Nucleic acid amplification of wheat 33391 genomic DNA produces an amplicon containing such diagnostic sequences. Also disclosed are isolated DNA molecules having a sufficient length of the transgene insert sequence and sufficient length of the wheat 33391 wheat genomic sequence to act as primer sequences for the production of the 33391 wheat diagnostic amplicon product.
Furthermore, methods for detecting the presence of DNA corresponding to wheat 33391 in a sample are disclosed. These methods include:
(a) contacting the sample containing the DNA with a primer arranged such that when used in a nucleic acid amplification reaction with genomic DNA from wheat 33391, it produces an amplicon that is diagnostic for wheat 33391;
(b) performing an amplification reaction, thereby providing an amplicon; and (c) detecting the amplicon.
According to another aspect, wheat detection kit 33391 is provided. The kit comprises at least one DNA sequence of sufficient length for polynucleotides complementary to SEQ ID NO: 5 or SEQ ID NO: 6, which DNA sequences are useful as primers or probes for which hybridize to DNA isolated from 33391 wheat or its progeny.
PL 214 848 B1
According to another aspect, there are provided methods of producing a wheat plant with improved glyphosate tolerance, the methods comprising the steps of:
(a) sexual crossing of a first parental line containing the pMON30139 construct which confers improved glyphosate tolerance with a second parental line which is intolerant to glyphosate, thereby producing a multiplicity of plant progeny; and (b) selecting a progeny plant that tolerates the use of glyphosate.
These methods are useful for introducing glyphosate tolerance into a variety of genetic backgrounds. Such methods may optionally include the further step of backcrossing the progeny of the plant with a second parent wheat line to produce a glyphosate tolerant wheat plant.
These as set out above and other aspects will become apparent from the following detailed description and attached figures. The attached figures show:
Figure 1. Map of the plasmid pMON30167
Figure 2. Map of the plasmid pMON42411
Figure 3. Map of the plasmid pMON30139
The following definitions and methods are provided to better define the present invention and to guide those skilled in the art to practice the present invention. Unless otherwise indicated, the terms are to be understood according to their conventional use by those skilled in the art in the relevant field. Definitions of terms common in molecular biology can also be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; I Lewin, Genes V, Oxford Jniversity Press: New York, 1994. The nomenclature used for DNA is as set out in 37CFR § 1.822.
As used herein, the term "wheat" means Triticum aestivum (including spring, winter and any wheat varieties) and other wheat species that can be grown from Triticum aestivum, including but not limited to durum wheat. spelled (Triticum spelta) and emmer wheat (Triticum diccocum). Also included are plants that are produced by conventional techniques using Triticum aestivum as a parent in a sexual cross with non-Triticum species (such as rye [Secale cereale]), including but not limited to triticale.
As used herein, the term "comprising" means "including but not limited to."
"Glyphosate" refers to N-phosphonomethylglycine and its salts. Glyphosate is the active ingredient in Roundup® herbicide (Monsanto Co, St. Louis, MO). Glyphosate Herbicide Treatment refers to treatment with Roundup®, Roundup Utra®, or any other formulation containing glyphosate. For the purposes of the present invention, the term "glyphosate" includes any herbicidally active form of N-phosphonomethylglycine (including any salt thereof) and other forms that result in the production of glyphosate anion in plants. Treatment with "glyphosate", unless otherwise stated, refers to treatment with the herbicide formulation Roundup®, Roundup Utra®. Plant transformation and regeneration in tissue culture use glyphosate or glyphosate salts. Whole plant studies use Roundup® or Roundup Utra®. Additional preparations with herbicidal activity containing N-phosphonomethylglycine or any salts thereof are included herein as glyphosate herbicide.
A transgenic "case" is produced by transforming plant cells with heterologous DNA, that is, with a nucleic acid construct that contains the transgene of interest, regenerating a population of plants obtained from inserting a transgene into the genome of that plant, and selecting individual plants characterized by an insert at a specific site in the genome.
The term "case" refers to the original transformant and the progeny of that transformant that contain heterologous DNA.
The term "case" also refers to the progeny produced by an external sexual cross between a transformant and other variants that contain heterologous DNA. Even after repeated backcrossing to the constant parent, the insert DNA and flanking DNA from the transformed parent are present in the progeny of the cross at the same chromosome site.
The term "case" also refers to DNA from the original transformant and its progeny containing the insert DNA and genomic flanking sequence immediately adjacent to the insert DNA that might be expected to be transferred to progeny that receive the insert DNA containing the transgene being sought, as evidenced by the cross. sex of one parental line,
This includes the inserted DNA (e.g., the original transformant and the offspring resulting from self-crossing) and a parental line that does not contain the inserted DNA.
The term "case" according to the invention includes wheat seed 33391 having ATCC Accession No. PTA-2347 and wheat plants grown from 33391 wheat and its progeny. A wheat plant that tolerates sufficient amounts of the glyphosate herbicide to control weeds in the field without affecting the wheat plants can be grown by a first sexual cross of the first wheat parent parent plant comprising the pMON30139 expression cassettes which confer improved tolerance to the use of the glyphosate herbicide and the second wheat parent plant with intolerance to glyphosate herbicide, whereby a variety of plants from the first progeny is produced; and then selecting a plant from the first progeny that is tolerant to the use of the glyphosate herbicide and self-crossing of the first progeny plants thereby producing plant diversity of the second progeny; and then selecting from the second progeny of plants that are tolerant to the glyphosate herbicide. These steps may additionally involve backcrossing the first-generation glyphosate-tolerant or second-generation glyphosate-tolerant plants with a second parental wheat plant or a third parental wheat plant, thereby producing a wheat plant that is tolerant to the use of the glyphosate herbicide. A crop containing 33391 wheat seeds or their offspring can be grown in the field and treated with a sufficient amount of glyphosate herbicide to control weeds without significantly affecting the wheat yield. A sufficient amount of glyphosate herbicide is about 8 ounces / acre or greater, 16 ounces / acre or greater, 32 ounces / acre or greater, or 64 ounces / acre or greater. Each glyphosate containing herbicide formulation can be used to control weeds in the cultivation of the 33391 wheat plant or its progeny.
It should also be understood that two different transgenic plants may also be mated to produce progeny that contain two independently segregating added, exogenous genes. Self-crossing of appropriate progeny can produce plants that are homozygous for both added and exogenous genes encoding the polypeptide of interest. Backcrossing of parent plants and out-crosing with non-transgenic plants as well as vegetative reproduction are also contemplated. Descriptions of other breeding methods that are commonly used for various traits and crops can be found in one of many publications, e.g., Fehr, in "Breeding Methods for Cultivar Develeopment, Wilcox J. Publisher, American Society of Agronomy, Madison WI (1987) in Entirely included in the description by quotation; Poehlman, JM (1987); Breeding Field Crops, 3rd edition. Van Nostrand Reinhold, NY, Knott, DR (1987); Included in their entirety by citation. The Application of Breeding Procedures to Wheat, pp. 419-427. In EG Heyne (publisher). In "Wheat and Wheat Improvement", Madison, WI, incorporated in its entirety by citation. Reverse cultures have been used to transfer genes of simple, innate, highly heritable traits to a desired homozygous cultivar or inbred line that is the return parent. The source of the trait to be transferred is called the donor parent. The resulting plant is expected to possess the attributes of the return parent (e.g. cultivar) and the desired trait transferred from the donor parent. After initial crossing, individual individuals having the donor parent phenotype are selected and re-crossed (backcross) with the return parent. The resulting parent should have the attributes of the return parent (e.g. cultivar) and the desired trait transferred from the donor parent.
The disclosed DNA molecules can be used as molecular markers in a marker-associated culture method (MAB). Such DNA molecules can be used in methods such as the markers AFLP, RFLP, RAPD, SNPs, and SSRs that identify genetically related agronomically useful traits as described in Walton, Seed World 22-29 (July, 1993), in its entirety incorporated by citations; Burlow and Blake Molecular Dissection of Complex traits, 13-29, Paterson Editors, CRC Press, New York (1988), incorporated in its entirety by citation). The improved glyphosate tolerance of the 33391 wheat plant can be marked in the progeny of a cross with the 33391 wheat plant and any other crop or variety using the MAB methods. DNA molecules are markers for such a trait and MAB methods well known in the art can be used to mark glyphosate tolerance in wheat where the 33391 wheat plant was a parent or ancestor.
A "probe" is an isolated nucleic acid to which is attached a conventionally detectable labeled or reporter molecule, e.g., a radioactive isotope, ligand, chemical agent.
Luminescent or enzyme. Such a probe is complementary to the strand of the target nucleic acid in the case of the present invention, to the genomic DNA strand of the wheat 33391 case (either from the wheat plant or from the sample containing DNA in this case). The disclosed probe includes not only deoxyribonucleic or ribonucleic acid, but also polyamides and other probe materials that bind specifically to a target DNA sequence and can be used to detect the presence of the target DNA sequence.
"Primers" are isolated nucleic acids that are fused into a complementary target DNA strand by hybridizing the nucleic acid to form a hybrid between the primer and the target DNA strand, followed by extension along the target DNA strand by a polymerase, e.g., by a DNA polymerase. Primer pairs or kits can be used to amplify a nucleic acid sequence, e.g. by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.
Probes and primers are at least 8 or more polynucleotides in length, at least 24 or more polynucleotides in length, at least 30 or more polynucleotides in length. These polynucleotides are useful as probes and primers that are of sufficient length to hybridize to a specific target sequence under stringent hybridization conditions. The disclosed probes and primers have full sequence similarity to a target sequence, although probes that are different from the target sequence and which remain capable of hybridizing to the target sequence may be determined by conventional methods.
Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd Edition, Vol. 1-3, Edited by Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 (hereinafter, Sambrook et al., 1989), incorporated in its entirety by citation; Current Protocols In Molecular Biology, edited by Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (updated periodically) (hereinafter Ausubel et al., 1992), incorporated in its entirety by cited herein; and Innis et al., PST Protocols: A Guide to ^ Methods and Applications, Academic Press: San Diego, 1990, in its entirety incorporated herein by reference. PCR-primer pairs can be derived from known sequences, for example, by the use of computer programs designed for these purposes, such as the Primer (Version 0.5, © 1991, Whitehead Institute for Biochemical Research, Cambridge, MA), wholly incorporated herein by reference.
The primers and probes based on flanking DNA and insert sequences disclosed herein may be used to confirm (and if necessary correct) the disclosed sequences by conventional methods, e.g., by re-cloning and sequencing such sequences.
The nucleic acid probes and primers disclosed herein hybridize under stringent conditions to a target DNA sequence. Any conventional hybridization and amplification method can be used to identify the presence of DNA from transgenic events in a sample.
The term "stringent conditions" is defined by a function with respect to hybridization of a nucleic acid probe to a target nucleic acid (i.e., with the particular nucleic acid sequence of interest) by a specific procedure discussed in Sambrook et al., 1989, 9.52-9. 55. See also Sambrook et al., 1989, 9.47-9.52, 9.56-9.58, incorporated herein by reference in its entirety; Kanahisa, (Nucl. Cid res. 12: 203-213, 1984, fully incorporated herein by citation); and Wetmur and Davidson, (U. Mol. Biol. 31: 349-370, 1988, incorporated herein by reference in its entirety.) Accordingly, the disclosed nucleic acid sequences may be used for their ability to selectively form duplex molecules with complementary stretched DNA fragments. Depending on the intended application, different hybridization conditions will be used to achieve different degrees of selectivity of the probe to the target sequence. For applications requiring high selectivity, once this will typically require the use of relatively stringent hybridization conditions to form hybrids, e.g., relatively weak salts and / or high temperature conditions such as those provided at about 0.02 M to about 0.15 M will be selected. NaCl at about 50 ° C to about 70 ° C. High stringency hybridization conditions, for example, are washing the hybridization filter at least twice with high stringency wash buffer (0.2 x SSC, 0.1% SDS, 65 ° C). Suitable stringency conditions that promote DNA hybridization and are known in the art to those skilled in the art include, for example, 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C followed by washing with 2.0 x SSC at about 50 ° C. and these conditions can be found in Current Protocols In Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. For example, the salt concentration in the washing step may be selected from a low stringency of about 2.0 x SSC at 50 ° C to a high stringency of about 0.2 x SSC at 50 ° C. Moreover, the temperature in the washing step may be increased
From low stringency conditions at room temperature, about 22 ° C, to high stringency conditions at about 65 ° C. Both the temperature and the salts can vary, or both the temperature or salt concentration can be kept constant while other variables are changed. Such selective conditions tolerate little, if any, mismatch between the probe and the pattern or target strand. Detection of DNA sequences by hybridization is well known to those skilled in the art and is taught in US Pat. US Am. Nos. 4,965,188 and 5,176,995 are exemplary hybridization analysis methods.
Regarding the amplification of a target nucleic acid sequence (e.g., by PCR) using a specific amplification primer pair, "stringent conditions" are conditions that allow the primer pair to hybridize only to that nucleic acid target sequence to which the primer has the corresponding wild-type sequence (or its complement) could bind to and preferentially produce amplicon - a unique amplification product.
The term "(target sequence) specific" indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample containing the target sequence.
As used herein, "amplified DNA" or "amplicon" means the nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a wheat plant derived from a sexual cross contains a transgenic event, genomic DNA from a wheat plant may be nucleic acid amplified using a primer pair containing a primer derived from a flanking sequence in the genome of that plant adjacent to the insertion site of the heterologous insertion. DNA and a second primer pair derived from the inserted heterologous DNA to produce, diagnostic for the presence of this case, amplicon. The amplicon has a length and sequence that is diagnostic for this case. Alternatively, the primer pair may be derived from a sequence flanking both insertion sites in order to generate an amplicon containing the entire insert.
Nucleic acid amplification can be completed by a variety of amplification methods known in the art, including polymerase chain reaction (PCR). Many amplification methods are known in the art and are described in, among others, US Pat. US Am. with Nos .: 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, Ed. Innis et al., Academic Press, San Diego, 1990. Any well known method for nucleic acid amplification can be used in the practice of the invention. The sequence of the heterologous DNA insert or flanking sequence from the case of wheat 33391, accession number PTA-2347 can be verified (and corrected if necessary) by amplifying such sequences from this case using primers derived from the sequences presented herein, using standard amplicon or cloned PCR DNA sequencing methods. DNA molecules.
The amplicon produced by these methods can be detected by a variety of techniques. Agarose gel electrophoresis and ethidium bromide staining is a common well known method for the detection of DNA amplicons. Another method is Genetic Bit Analysis (Nikiforov, et al., Nucleic Acid Res. 22: 4167-4175, 1994) where the DNA of the oligonucleotide is designed to overlap with both the adjacent flanking genomic DNA sequence and the insert DNA sequence. This oligonucleotide is immobilized in the wells of the microtiter plates. After PCR of the target region (using one primer in the insert sequence and one in the adjacent genomic flanking sequence), a single-stranded PCR product can be hybridized to the immobilized oligonucleotide and serve as a template for a monobasic extension reaction using DNA polymerase and labeled ddNTPs specific for the expected next base . Reading may be fluorescence or ELISA based. The signal indicates the presence of an insert / flanking sequence resulting from successful amplification, hybridization and monobasic extension.
An additional method is the Pyrosequencing technique as described by Winge (Innov. Pharma. Tech. 00: 18-24, 2000). In this method, the oligonucleotide is designed to overlap the junction - the adjacent genomic DNA sequence and the DNA insert. This oligonucleotide is hybridized to a single-stranded PCR product from the target region (one primer in the insertion sequence and one in the flanking genomic sequence) and incubated in the presence of DNA polymerase, ATP, sulfurylase, luciferase, aperase, adenosine-5-phosphate and luciferin. DNTPs are added individually and the light signal resulting from the incorporation is measured. The light signal indicates the presence of the amplification transgene / flanking sequence by successful hybridization and single or multi base extension.
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Fluorescence Polarization as described by Chen et al. (Genome Res. 9: 492-498, 1999) is a method that can be used to detect an amplicon. Using this method, the oligonucleotide is designed to mesh with the genomic junction flanking the inserted DNA. The oligonucleotide is hybridized to the single-stranded PCR product from the target region (one primer in the insert DNA and one in the flanking and genomic DNA sequences) and incubated in the presence of DNA polymerase and fluorescently labeled ddNTP. A monobasic extension leads to ddNTP incorporation. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarity indicates the presence of a transgene / flanking sequence resulting from successful amplification, hybridization and monobase extension.
Tagman® (PE Applied Biosystems, Foster City, CA) is described as a method of detecting and quantifying the presence of a DNA sequence and is fully understood from the manufacturer's instructions. Briefly, a FRET oligonucleotide probe is designed that meshes with the junction of genomic flanking and insert DNA. The FRET probe and the PCR primers (one primer in the DNA insert sequence and one in the genomic flanking sequence) are cyclized in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe breaks and releases a fluorescent molecule from the cooling molecule on the FRET probe. A fluorescent signal indicates the presence of a flanking / transgene sequence resulting from successful amplification and hybridization.
Molecular Beacons have been described for use in sequence detection as in Tyangi et al. (Nature Biotech. 14; 303-308, 1996). Briefly, a FRET oligonucleotide probe is designed that meshes with the junction of the genomic flanking and insert DNA. The unique design of the FRET probe leads to it having a secondary structure that keeps fluorescent and cooling molecules in close proximity. The FRET probe and the PCR primers (one primer in the insert DNA sequence and one in the genomic flanking sequence) are cyclized in the presence of the thermostable dNTPs polymerase. After successful amplification, hybridization of the FRET probe to the target sequence removes the probe secondary structure and spatially separates the fluorescent and cooling molecules. A fluorescence signal is obtained. A fluorescent signal indicates the presence of a flanking / transgene sequence resulting from successful amplification and hybridization.
In order to illustrate some preferred embodiments of the invention, the following examples are included. It should be understood by one of ordinary skill in the art that the techniques disclosed in the examples which follow the approaches shown by the inventors will work well in the practice of the invention and thus can be considered as exemplary of the preferred modes of embodiment. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made to certain disclosed forms and still obtain such and similar results without departing from the spirit and scope of the invention.
Example I.
Wheat transgenic plants are generated by Agrobacterium dependent transformation of wheat embryos by the method of Cheng et al. (Plant Physiol. 115: 971-980, 1997) using binary vectors and modifying the glyphosate selection conditions of Zhou et al. (Plant Cell Rep. 15: 159 -163,1995). Other methods of transforming wheat are known to those skilled in the art, such as gene weapons or particle bombardment, and can be used to insert expression cassettes into the genome of wheat cells. The T-DNA of pMGN30139 (Figure 3) contains two expression cassettes that together confer a high degree of tolerance to the glyphosate herbicide. The first transgene expression cassette contains rice actin promoter DNA sequences and an intron (P-Os.Act1 and I-Os.Act1, US Patent 5,641,876, fully incorporated herein by reference) operably linked to the DNA sequence encoding Arabidopsis thaliana Chloroplast transit peptide EPSPS (TS-At.EPSPS: CTP2, Klee et al., Mol. Gen. Genet. 210: 47-442, 1987, in its entirety incorporated herein by reference), operably linked to a DNA sequence encoding glyphosate resistant 5-enolopyruviloshikimato-3-phosphate synthase (EPSPS) isolated from Agrobacterium tumefaciens (AGRTU.aroAgene, US Patent No. No. 5,633,435, incorporated by reference in its entirety), operably linked to the nopaline synatase transcription terminator (T-AGRTU. Nos, Fraley et al., Proc. Natl. Acad. Sci. USA 80: 4803-4807, 1983, incorporated by reference in its entirety). The second transgene expression cassette contains cauliflower mosaic virus 35S promoter DNA sequences (P-CaMV.35S: en, Kay et al., Science 236: 1299-1302, 1987; US Patent 5,164,316 in its entirety incorporated herein by reference) , operably linked to the DNA sequence of the Zea mays Hsp70 intron (I-Zm.Hsp70, U.S. Patent No. No. 5,424,412, incorporated by reference in its entirety), an operably linked DNA sequence encoding the Arabidopsis thaliana EPSPS chloroplast transit peptide sequence (TS-At.EPSPS,
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Klee et al., Mol. Gen. Genet. 210: 47-442. 1987), operably linked to the coding sequence for glyphosate resistant 5-enol-pyruviloshikimato-3-phosphate synthase (EPSPS) isolated from Agrobacterium tumefaciens sp. Strain CP4 (AGRTU.aroAgene, U.S. Patent No. 5,633,435, in its entirety included in here by citation), operably linked to the nopaline synatase transcription terminator (T-30 AGRTU.nos, Fraley et al., Proc. Natl. Acad. Sci. USA 80: 4803-4807, 1983.
pMON30167 (Figure 1) is a single expression cassette identical to the expression caste of the first pMON30139 transgene as described above. pMON42411 (Figure 2) is a single expression cassette identical to the second expression cassette pMGN30139 as described above.
After incubation of wheat cells with Agrobacterium cells containing the pMON42411, pMON30167 and pM030139 constructs, glyphosate tolerant wheat calluses were selected on media containing 2 mM glyphosate for 1 week followed by transfer to 0.1 mm glyphosate differentiation media for 2 weeks and finally transferred for regeneration medium with 0.02 mM glyphosate + 0.1 μΜ aromatic acids.
Two hundred and eighty-four wheat cases were produced from the transformation with pMON42411, pMON30167 and pM030139. These plants which were derived from pMO30139 and pMON30167 were sprayed once at 64 oz / acre with glyphosate herbicide. Selection of transformed wheat plants with one expression cassette pMON42411 and pMON30167 resulted in a small percentage (1.4% 13.2%, respectively) of wheat plants with both vegetative and reproductive tolerance. Only 3/134 plants from these constructs had an acceptable level of tolerance to the glyphosate herbicide. In contrast, transformed wheat plants containing the pMON30139 dual expression cassette produced a high percentage (16%) of plants with both vegetative and reproductive tolerance (25/100).
Wheat case 33391 (henceforth referred to as wheat plant 33391 or wheat 33391 includes all parts of the wheat and seeds of this plant) was selected from 150 transgenic wheat cases generated from the transformation of pMON30139. Twenty-four were selected from the population that demonstrated improved vegetative and reproductive tolerance of glyphosate. A further evaluation of these 24 cases was performed for the agricultural project and the presence of a single intact insert. 33391 wheat was selected from this case population. Greenhouse and field evaluations of 33391 wheat and 33391 wheat-derived progeny indicated that this transgenic insert gave glyphosate tolerance beyond the commercial specification for full vegetative and reproductive tolerance of 340 g glyphosate / acre (840 g glyphosate / hectare; 32 oz Roundap ' u Ultra / acre) with a two-fold safety margin when used in the 3-5 leaf stage.
Table 1
Comparison of the efficacy of single and dual expression cassettes for glyphosate tolerance in wheat
<td>pMON #</td><td># tested cases</td><td># cases with vegetative tolerance</td><td># weg. tolerant cases with reproductive tolerance</td>
<td> 42411</td><td> 71</td><td> 26</td><td> 1(1,4%)</td>
<td> 30167</td><td> 63</td><td> 4</td><td> 2(3,3%)</td>
<td> 30139</td><td> 150</td><td> 104</td><td> 24(16%)</td>
Example 2
Isolation of the corresponding flanking genomic sequence is possible by a number of methods known to those skilled in the art (for example, using ligated adapters and nested PCR as described in the Genome Walker ™ kit (Clonetech laboratories, Inc, Palo Alto, CA). Wheat genomic DNA. 33391 was isolated by CTAB purification (Rogers et al., Plant Mol Biol 5: 69-76, 1985). Reagents are commercially available (see, for example, Sigma Chemical Co., St. Louis, MO). Genomic libraries for amplification were prepared according to the manufacturers' instructions (Genome Walker ™, Clonetech Laboratories, Inc, Palo Alto, CA). In a separate reaction, genomic DNA was digested with restriction enzymes overnight at 37 ° C with the following blunt ended endonucleases: DraI, EcoRV, PvuII, Scal, and Stul (CloneTech Laboratories, Inc., Palo Alto, CA.). was extracted with phenol: chloroform, DNA was precipitated by adding ethanol to the aqueous phase, pelleted by centrifugation, and then resuspended in Tris-EDTA buffer (10 mM Tris-HCl, pH 8.0, 1 mM RDTA). Purified genomic fragments
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The blunt ended DNA was then ligated to the Genome Walker ™ adapters according to the manufacturer's protocol. After these adapters were ligated to genomic DNA fragments, each reaction was heat treated (70 ° C for 5 minutes) to stop the reaction and then diluted 10-fold with Tris EDTA buffer. One μΐ of each representative ligation was then amplified in a 50 μΐ reaction according to the manufacturer's recommended protocol using an adapter-specific oligonucleotide (supplied by the manufacturer) and a transgenic-specific 33391 wheat oligonucleotide, such as SEQ ID NO: 1, which splits past the 5 'end of P- Os.Act1. The PCR mix contained 1 µl of the appropriate adapter ligated library, 1 µl, 10 µl of the Genome Walker ™ adapter, AP1 primer supplied by the manufacturer (5'GTATAlCGACTCACTATAGGGC3 ', SEQ ID NO: 11), 1 µl 10 µΜ of the transgene specific oligonucleotide (SEQ ID NO: 1), 1 μΐ 10 mM deoxyribonucleotides, 5 μΐ PCR 10Χ buffer containing MgCl<sub>2</sub>, 0.5 μΐ (2.5 units) Tag DNA polymerase (Borhringer Mannheim Bichemicals, Indianapolis, IN), and H<sub>2</sub>Up to 50 μΐ. The PCR reaction was performed in a thermocycler using the calculated control temperature and the following cycle conditions: 1 cycle at 94 ° C for 1 minute; 7 cycles (94 ° C for 2 seconds, 70 ° C for 3 minutes); 37 cycles at (94 ° C for 2 seconds, 65 ° C for 3 minutes); 1 cycle at 65 ° C for 10 minutes. One μΐ of each primary reaction was then amplified in a second amplification using an internal adapter-specific oligonucleotide (supplied by the manufacturer) and an internal transgene-specific oligonucleotide such as SEQ ID NO: 2 which binds to P-Os.Act1 upstream of the primer used in the reaction primary. The repeat PCR mix contained 1 μΐ of the corresponding primary PCR products, 1 μΐ 10 μΜ Genome Walker ™ internal adapter, AP2 primer supplied by the manufacturer (5'ACTATAGGGCACGCGTGGT3 ', SEQ ID NO: 12), 1 μΐ 10 μΜ transgeno-specific internal oligonucleotide ( SEQ ID NO: 2), or 10 μΜ deoxyribonucleotides, 5 μΐ 10XPCR of buffer containing MgCl<sub>2</sub>, 0.5 μΐ (2.5 units) Tag DNA polymerase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and H<sub>2</sub>Up to 50 μΐ. PCR reactions were again performed in a thermocycler using the calculated control temperature and the following cycling conditions: 1 cycle at 94 ° C for 1 minute; 7 cycles (94 ° C for 2 seconds, 70 ° C for 3 minutes); 31 cycles at (94 ° C for 2 seconds, 65 ° C for 3 minutes), 1 cycle at 65 ° C for 10 minutes. The PCR products, representing the 5 'regions junction between the 33391 transgenic wheat insert and the flanking genomic adjacent sequence, were then purified by agarose gel electrophoresis and then isolated from agarose matrix using the QIAquick Del Extraction Kit (catalog # 28704, Qiagen Inc., Valencia). , CA) and then cloned directly into the pGEM-T Easy vector (Catalog # A1360, Promega, Madison, WI). The identity of the cloned products was confirmed by DNA sequence analysis (ABI Prism ™ 337, PE Biosystems, FosterCity, CA and DNASTAR sequence analysis software, DNASTAR Inc, Madison, WI).
Similarly, the 3 'flanking genomic DNA sequence was amplified and cloned using internal specific gene primers, such as SEQ ID NO: 3, and SEQ ID NO: 4, which bind to the T-nos transcription terminator. Two t-nos transcriptional terminators are present in the wheat transgenic / genomic insert 33391, one internal to the construct and one at the 3 'end of the construct adjacent to the wheat genomic sequence. The PCR products produced by this reaction were sequenced and the DNA sequence that bridged the junction between the transgene and the flanking genomic was distinguished from the internal T-nos products by comparison with known elements of the genetic sequence of the pMON 30139 construct.
The wheat genomic sequence flanking both insertion sites of the transgene in the wheat genome was determined for wheat 33391 by sequencing the amplification products derived from Genome Walker ™ and aligning to the known transgene sequence. A sequence of 339 bp around the insert site was marked at the 5 'end of the transgene insert site. This segment was the 257 (bp) wheat genomic sequence (nucleotide bases 258-350 of SEQ ID NO: 5) and 49 bp of the 5 'end of the Act1 rice actin promoter (nucleotide bases 251-399 of SEQ ID NO: 5). Similarly, DNA sequence was determined for the 431 bp segment flanking the insertion junction (SEQ ID NO: 6), starting with 32 bp T-nos transcription terminator sequence (nucleotide bases 1031 of SEQ ID NO: 6), 68 bp vector backbone sequence (bases nucleotide 33-100) and the 331 bp-terminating wheat genomic sequence flanking the transgene insertion site (nucleotide bases 101-431 of SEQ ID NO: 6). Identification of wheat 33391 was performed by PCR amplification of the region of the transgene / genomic insert using one primer from the transgene sequence and another primer from the wheat genomic flanking sequence.
The transgenic / genomic region of the 5 'insert was confirmed by PCR amplification of the DNA amplicon that it was unique to wheat 33391. This identification was demonstrated from the amplicon
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PCR generated by primer 5 (SEQ ID NO: 7) and primer 6 (SEQ ID NO: 8). Additional primer sequences can be synthesized using the DNA sequence shown in SEQ ID NO: 5, which will generate amplicons with a DNA length different than that generated by primer 5 and primer 6, but still diagnostic for wheat 33391 and its progeny. It is within the ordinary knowledge of a plant molecular biologist to select the DNA primer sequences from SEQ ID NO: 5 and to select hybridization stringency conditions to generate amplicon. Similarly, one skilled in the art will select a primer DNA sequence of SEQ ID NO: 6 which will generate diagnostic amplicons for wheat 33391. It is within the scope of the invention that DNA primer sequences derived from SEQ ID NO: 5 and SEQ ID NO: 6 are useful for isolating additional genomic DNA molecules from wheat 33391 plants, seeds, and plant parts by the methods disclosed herein or by methods known in the art. the state of the art in plant molecular biology. These additional wheat genomic DNA molecules may be disclosed in SEQ ID NO: 5 and SEQ ID NO: 6 useful as a primer or probe. These additional wheat genomic DNA molecules can be used as molecular diagnostic markers for wheat 33391.
The DNA sequences that span the junction region of the wheat 33391 genomic DNA and the pMON30139 DNA insert contained in SEQ ID NO: 5 and SEQ ID NO: 6 can be used as probes in a hybridization reaction to identify wheat-derived DNA 33391. For example, a DNA molecule useful as a probe of SEQ ID NO: 5 could include the nucleotide sequence of position 245-270 or the complement thereof; A DNA molecule useful as a probe of SEQ ID NO: 6 could include the nucleotide sequence of position 87-113 or the complement thereof. One skilled in the art can select nucleotide sequences shorter or longer than those described that span the splice region and are useful as specific DNA probes or primers for wheat 33391 under stringent hybridization conditions.
The PCR reaction conditions (Table 2) and the quality of the extracted wheat 33391 genomic DNA are confirmed by the production of amplicon by primer 7 (SEQ ID NO: 9) and primer 8 (SEQ ID NO: 10) and represent an approximately 400 bp DNA fragment from the carboxylase gene wheat acetyl CoA (Acc), a single copy of an endogenous gene in the wheat genome. Controls for these analyzes should include the 33391 Wheat Positive Control, and the Non-33391 Wheat Negative Control, and the Negative Control that contains wheat non-standard DNA as shown in Table 2. The 33391 wheat amplicon test may be performed by the use of a Stratagene Robocycler, MJ Engine, Perkin-Eimer 9700 or a Mastercycler Gradient thermal cycler as shown in Table 3, or by methods and apparatus known to one skilled in the art.
Table 2
PCR procedure and reaction mix to confirm the wheat 33391 transgenic / genomic junction region
<td>Stage</td><td>Reagent</td><td>Quantity</td><td>Comment</td>
<td> 1</td><td> 2</td><td> 3</td><td> 4</td>
<td> 1</td><td>Nuclease-free water</td><td>Added to a final volume of 20 μl</td><td> -</td>
<td> 2</td><td>10X reaction buffer (with MgCb)</td><td>2.0 pi</td><td>1X buffer concentration, 1.5mM final MgCl2 concentration</td>
<td> 3</td><td>10 mM solution of dATP, dCTP, dDTP and dTTP</td><td>0.4 pl</td><td>200 μΜ final concentration of each dNIP</td>
<td> 4</td><td>Primer 5 (SEQ ID NO: 7) (resuspended in 1X TE buffer or nuclease free water to a concentration of 10 μΜ)</td><td>0.4 pl</td><td>0.2 μΜ final concentration</td>
<td> 5</td><td>Primer 6 (SEQ ID NO: 8) (resuspended in 1X TE buffer or nuclease free water to a concentration of 10 μΜ)</td><td>0.4 pl</td><td>0.2 μΜ final concentration</td>
<td> 6</td><td>Primer 7 (SEQ ID NO: 9) (resuspended in 1X TE buffer or nuclease free water to a concentration of 10 μΜ)</td><td>0.2 pl</td><td>0.1 μΜ final concentration</td>
PL 214 848 B1 cont. table 2
<td> 1</td><td> 2</td><td> 3</td><td> 4</td>
<td> 7</td><td>Primer 8 (SEQ ID NO: 10) (resuspended in 1X TE buffer or nuclease free water to a concentration of 10 μΜ)</td><td>0.2 pl</td><td>0.1 μΜ final concentration</td>
<td> 8</td><td>RNase and DNase free (500 ng / ml)</td><td>0.1 pl</td><td>50 ng / reaction</td>
<td> 9</td><td>REDTaq DNA polymerase</td><td>1.0 pl (it is recommended to change the pipette before the next step)</td><td>1 unit / reaction</td>
<td> 10</td><td>Extracted DNA (standard): • analyzed sample • individual leaves • leaf pool (maximum 50 leaves / pool) • Negative control • Negative control • Positive control</td><td>• 10-200 ng of genomic DNA • 200 ng of genomic DNA • 50 ng wheat genomic DNA (not wheat 33391) • without DNA template • 50 ng of 33391 genomic DNA</td><td></td>
Table 3
Suggested PCR parameters for different thermal cyclers *
<td>Cycle number</td><td>Settings: Stratagene Robocycler</td>
<td> 1</td><td>94 ° C 3 minutes</td>
<td> 38</td><td>94 ° C 1 minute 63 ° C 1 minute 72 ° C 1 minute and 30 seconds</td>
<td> 1</td><td>72 ° C 10 minutes</td>
<td>Cycle number</td><td>Settings: MJ Engine or Perkin-Elmer 9700</td>
<td> 1</td><td>94 ° C 3 minutes</td>
<td> 38</td><td>94 ° C 10 seconds 63 ° C 30 seconds 72 ° C 1 minute</td>
<td> 1</td><td>72 ° C 10 minutes</td>
<td>Cycle number</td><td>Settings: Eppendorf Mastercycler Gradient</td>
<td> 1</td><td>94 ° C 3 minutes</td>
<td> 38</td><td>94 ° C 15 seconds 63 ° C 15 seconds 72 ° C 1 minute</td>
<td> 1</td><td>72 ° C 10 minutes</td>
Mix gently and if necessary add 1-2 drops of mineral oil on top of each reaction (not on the hot top of the thermal cycler).
Perform PCR on a Stratagene Robocycler, MJ Engine, Perkm-Elmer 9700 or Eppendorf Mastercycler Gradient thermal cycler using the following cycling parameters.
Note: The MJ Engine or the Eppendorf Mastercycler Gradient thermal cycler should be started in a calculated manner. Uniform speed operation of the Perkin-Elmer 9700 thermal cycler is set to maximum.
PL 214 848 B1
Example 3
The expression of glyphosate-resistant EPSPS (CP4 EPSPS) proteins from aroA: CP4 can be detected by immunological methods from plant tissue extracts (Rogan et al., Food Control 10: 407-414, 1999, in its entirety herein by citation). Immunological methods such as Western blots, test strips and an enzyme-linked immunosorbent assay (ELISA) have been used to accurately detect the expressed proteins from the aroA: CP4 gene contained in an expression vector transformed into plants. Reagents that contain polyclonal and monoclonal antibodies specific for EPSPS CP4 are commercially available from Strategie Diagnostics (Newark, DE). CP4 EPSPS can be detected from protein extracts of the 33391 wheat plant, plant parts and seeds by immunological methods including ELISA.
An ELISA procedure that uses 100 ng of an anti-CP4 EPSPS monoclonal antibody dissolved in 100 µl of 0.05 M sodium bicarbonate buffer, pH 9.6, is absorbed in a microtiter well overnight at 4 ° C. The well is washed with 0.05% Tween-20 phosphate salt buffer, pH 7.4 (PBS-T). Tissue is homogenized in phosphate buffered salt with a mortar and pistyl or other suitable tissue disintegrator. The homogenate is applied to a microtiter well and incubated for approximately 2 hours at 37 ° C. The well is washed three times with PBS-T. In one method, the second order antibody, purified rabbit anti-CP4 EPSPS is dissolved to a sufficient level to provide specific binding to the CP4 EPSPS protein and incubated at 37 ° C for approximately 1 hour. In a second approach, a second-order goat anti-CP4 EPSPS antibody is used. Biotin-conjugated anti-rabbit IgG or anti-goat IgG (Sigma Corp, St. Louis MO) is added to the well (1: 40,000 dilution in PBS) and incubated at 37 ° C for 30 minutes. The sump is washed three times with PBS-T. Horseradish peroxidase conjugated NeutrAvidin is diluted 1: 10,000 using a StabilZyme HRP stabilizer (SurModics, Eden Prairie, MN) and incubated at 37 ° C for 15 minutes. The well is washed three times with PBS-T. The TMB subsirate (Krikergard and Perry, Gathersburg, MD) is added over 10 minutes and then the reaction is quenched with 3M phosphoric acid. The well is read in a microtiter plate reader at 450 nm and a reference wavelength of 650 nm. This method is an example of a suitable ELISA test for the detection of CP4 EPSPS, and not only the ELISA method can be used to detect CP4 EPSPS, and the skilled person familiar with the issues of ELISA tests will know the variations of this method that can be intended to provide detection tests specific and sensitive enough for the detection of CP4 EPSPS in plant tissue extracts.
The ELISA from the fodder wheat field 33391 contains on average 58.2 ± 8.4 μg / g, with a range of 45.5 to 72.4 μg / g CP4 EPSPS protein based on fresh tissue weight (fwt), while the non-transgenic control field with seed had no detectable CP4 EPSPS protein level above the detection limit by ELISA at 0.9 µg / g fwt. Wheat grain tissue 33391 contains an average level of 12.6 ± 2.5 μg / g, with a range of 9.5 to 17.6 μg / g CP4 EPSPS protein per fresh tissue (fwt), while non-transgenic control feed does not had a detectable level of CP4 EPSPS protein above the limit of detection by ELISA at 0.1 µg / g fwt. ELISA or other immunological methods can be used as diagnostic assays for the detection of CP4 EPSPS for wheat 33391, while the 33391 wheat progeny is only USDA registered glyphosate tolerant wheat expressing the CP4 EPSPS protein.
The 33391 wheat deposit to Monsanto Company disclosed in this specification and referenced in the appended claims has been made in accordance with the Budapest Agreement for the Recognition of Microbial Deposits, at the American Standard Culture Collection (ATCC), 1081 University Boulevard, Manassas, Va. 20110. Access number - PTA-2347. This deposit will be held with the custodian for 30 years, or 5 years after the last request, or for the effective term of the patent, whichever is longer and will be replaced if necessary during that period.
By illustrating and describing the main principles of the present invention, it should be apparent to one skilled in the art that the invention may be modified in layout and detail without departing from these principles. All modifications are reserved within the spirit and scope of the appended claims.
PL 214 848 B1
All publications and published patent documents cited in this specification are incorporated herein by reference to the same extent as any individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Contents7
7 sheets
Sheet 1 Sheet 2 Sheet 3 Sheet 4 Sheet 5 Sheet 6 Sheet 7
20 members in 9 offices
Priority claims8
| Document | Office | Kind | Date |
|---|---|---|---|
| 23665300 | United States of America | P | |
| 23676200 | United States of America | P | |
| 0129902 | United States of America | W | |
| 60236762 | – | – | – |
| 60236653 | – | – | – |
| US20000236653P | – | – | – |
| US20000236762P | – | – | – |
| WO2001US29902 | – | – | – |
Members20
| Document | Office | Kind | |
|---|---|---|---|
| CA2420406A1 | Canada | A1 | |
| WO0227004A2 | World Intellectual Property Organization (WIPO) | A2 | |
| AU9304401A | Australia | A | |
| US2002062503A1 | United States of America | A1 | |
| WO0227004A3 | World Intellectual Property Organization (WIPO) | A3 | |
| EP1322773A2 | European Patent Office (EPO) | A2 | |
| CZ2003629A3 | Czechia | A3 | |
| AR030812A1 | Argentina | A1 | |
| US6689880B2 | United States of America | B2 | |
| ZA200301081B | South Africa | B | |
| US2004133939A1 | United States of America | A1 | |
| US2004133940A1 | United States of America | A1 | |
| PL365885A1 | Poland | A1 | |
| US7071325B2 | United States of America | B2 | |
| AU2001293044B2 | Australia | B2 | |
| US7268274B2 | United States of America | B2 | |
| CZ300073B6 | Czechia | B6 | |
| AR069294A2 | Argentina | A2 | |
| PL214848B1This record | Poland | B1 | |
| CA2420406C | Canada | C |
1 legal event, as the office reported them to INPADOC
Events
| Event | Code | |
|---|---|---|
| Rectifications of patent specificationRECP | RECP |
Numbers
- Publication
- 214848
- Publication, DOCDB
- 214848
- Publication, EPODOC
- PL214848B
- Application
- 365885
- Application, DOCDB
- 36588501
- Application, EPODOC
- PL20010365885
Titles2
- English
- GLYPHOSATE TOLERANT WHEAT PLANT 33391 AND COMPOSITIONS AND METHODS FOR DETECTION THEREOF
- Polish
- Sposób wytwarzania tolerancyjnej na glifosat rosliny pszenicy, plodna tolerujaca glifosat roslina pszenicy, nasiono pszenicy, para czasteczek DNA, sposób wykrywania obecnosci diagnostycznej czasteczki DNA, sposób wykrywania sekwencji zawierajacej okreslone nukleotydy, sposób wytwarzania rosliny pszenicy z tolerancja glifosatu, zestaw do wykrywania DNA, nasiono transgenicznej pszenicy, roslina pszenicy wytworzona przez hodowle nasion, sposób wytwarzania rosliny pszenicy tolerujacej stosowanie glifosatu, sposób selektywnego kontrolowania chwastów na polu zawierajacym uprawe pszenicy, czasteczka DNA, roslina pszenicy tolerujaca glifosat, roslina zdolna do wytwarzania diagnostycznego amplikonu, nasiono, komórka
Classification
- CPC, 3
- C12N9/1092
- C12N15/8275
- C12Y205/01019
- IPC, 7
- C12N15 29
- A01H5 10
- A01H5 12
- C12N9 10
- C12N15 10
- C12N15 82
- C12N15 84
