Regulation of quinolate phosphoribosyl transferase expression in tobacco plants

Regulation of quinolate phosphoribosyl transferase expression

FIELD OF THE INVENTION

The present invention relates to plant quinolate phosphoribosyl transferase (QPRTase) and DNA encoding this enzyme. In particular, the present invention relates to the use of DNA encoding quinolate phosphoribosyl transferase for the production of genetically modified nicotine-containing transgenic plants, and plants so produced.

BACKGROUND OF THE INVENTION

The production of tobacco with reduced nicotine content is of interest, particularly because of the narcotic nature of nicotine. In addition, particularly low nicotine or nicotine-producing tobacco plants are attractive recipients for transgenes expressing commercially valuable products such as pharmaceuticals, cosmetic components or nutritional supplements. There are various processes designed to remove nicotine from tobacco. However, most of these processes remove other ingredients from tobacco in addition to nicotine, thus adversely affecting tobacco. Classical crop production methods have produced tobacco plants that have a lower (about 8%) nicotine content than wild-type tobacco plants. It is desirable that tobacco plants and tobacco have even lower nicotine levels.

One approach to reducing the amount of biological product is to reduce the amount of enzyme required in the biosynthetic pathway that leads to product formation. If the affected enzyme is naturally present in a rate-limiting amount (compared to other enzymes required in the metabolic pathway), any reduction in the amount of enzyme will reduce the production of the final product. If the natural enzyme content is not rate limiting, the amount of this enzyme in the cell must be reduced to a rate limiting level to reduce the productivity of the synthesis pathway. Conversely, the natural content of the enzyme is

EE 04595 B1 reaction rate limiting, any increase in enzyme activity increases the productivity of the end product of the biosynthetic pathway.

Nicotine is first formed at the roots of a tobacco plant and then transported to the leaves where it is stored (Tso, Physiology and Biochemistry of Tobacco Plants, 233-34, Dowden, Hutchinson & Ross (Eds.), Stroudsburg, Pa. 1972). A mandatory step in nicotine biosynthesis is the formation of nicotinic acid from quinolinic acid, which is catalyzed by the enzyme quinoline phosphoribosyl transferase (QPRTase). QPRTase is a rate-limiting enzyme in the pathway that produces nicotinic acid required for the synthesis of nicotine in a tobacco plant (Feth et al., 1986, Planta 168, 402-07; Wagner et al., The Regulation of Enzyme Activities). the Nicotine Pathway in Tobacco, Physiol. Plant., 68, 667-72 (1986). U.S. Patent Nos. 5,369,023 and 5,260,205 (Nakatani and Malik) suggest modification of nicotine content in tobacco plants by antisense regulation of putrescine methyltransferase (PMTase) expression. Wahadi and Malik PCT Patent Application WO 94/28142 describes DNA encoding PMT and the use of PMT and sense and antisense PMT constructs.

SUMMARY OF THE INVENTION

A first aspect of the present invention is an isolated DNA molecule comprising SEQ ID NO: 1, DNA sequences encoding the enzyme SEQ ID NO: 2, DNA sequences which hybridize to such DNA and encode the enzyme quinolate phosphoribosyl transferase and DNA sequences different from those described above. DNA has a degenerate genetic code. A peptide encoded by such DNA is another aspect of the invention.

Another aspect of the present invention is a DNA construct comprising a plant cell-active promoter and a DNA fragment encoding the enzyme quinolate phosphoribosyl transferase

EE 04595 B1 and is operably linked to the promoter. The DNA encoding the enzyme may be in sense or antisense orientation.

An aspect of the present invention is a method of producing transgenic plant cells with reduced expression of quinolate phosphoribosyl transferase (QPRTase), which provides plant cells of the type expressing quinolate phosphoribosyl transferase by transforming the plant cell with an exogenous DNA construct comprising a promoter and a DNA containing mRNA.

An aspect of the present invention is a transgenic plant of the species Nicotiana genus which has reduced expression of quinolate phosphoribosyl transferase (QPRTase) compared to a non-transformed control plant. Cells of such a plant contain a DNA construct comprising a segment of the DNA sequence encoding the plant quinolate phosphoribosyl transferase mRNA.

Another aspect of the present invention is a method of reducing expression of a quinolate phosphoribosyl transferase gene in a plant cell by growing such transformed plant cells to contain exogenous DNA wherein the transcribed strand of exogenous DNA is complementary to endogenous cellular quinolate phosphoribosyl transferase mRNA. Complementary strand transcription decreases expression of the endogenous gene of quinolate phosphoribosyl transferase.

Another aspect of the present invention is a method of producing a tobacco plant having reduced nicotine content in the leaves by growing tobacco plants with cells containing an exogenous DNA sequence in which the transcribed strand of the exogenous DNA sequence is complementary to endogenous quinolate phosphoribosyl transferase mRNA.

Another aspect of the present invention is a method of producing transgenic plant cells having higher expression of quinolate phosphoribosyl transferase (QPRTase) by transforming a quinolate phosphoribosyl transferase expressing plant cell with an exogenous DNA construct containing quinolate phosphoribosyl transferase.

Another aspect of the present invention is the Nicotiana transgenic plant with increased expression of quinolate phosphoribosyl transferase (QPRTase), wherein the cells of the transgenic plant contain an exogenous DNA sequence encoding the plant quinolate phosphoribosyl transferase.

Another aspect of the present invention is a method of increasing expression of a quinolate phosphoribosyl transferase gene in plant cells by growing plant cells transformed to contain quinolate phosphoribosyl transferase encoding exogenous DNA.

Another aspect of the present invention is a method of producing tobacco plants having leaves with increased nicotine content by growing tobacco plants whose cells contain an exogenous DNA sequence encoding a functional quinolate phosphoribosyl transferase in cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the nicotine biosynthetic pathway. Nic1 and Nic2 are known to be regulated enzymes QPRTase (quinolate phosphoribosyltransferase) and PMTase (putrescine methyltransferase).

Figure 2A shows the nucleic acid sequence of the NtQPT1 cDNA (SEQ ID NO: 1)_? wherein the coding sequence (SEQ ID NO: 3) is in capital letters.

Figure 2B shows the deduced amino acid sequence of the NtQPT1 cDNA encoded tobacco QPRTase (SEQ ID NO: 2).

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Figure 3 shows the deduced NtQPT1 amino acid sequence and similar sequences for Rhodospirillum rubrum, Mycobacterium leprae, Salmonella tuphimurium, Escherichia coli, human and Saccharomyces cerevisiae.

Figure 4 shows the results of complementation of an Escherichia coli mutant lacking quinolate phosphoribosyl transferase (TH265) with the NtQPT1 cDNA. Cells were transformed with the NtQPT1 expression vector. Growth of transformed TH265 cells expressing NtQPT1 in minimal nicotinic acid-free medium indicated that NtQPT1 encodes QPRTase.

Figure 5 compares nicotine content and NtQPT1 mRNA content in steady state tobacco Nic1 and Nic2 mutants: wild-type Burley 21 (Nicl / Nicl Nic2 / Nic2), Nicl-Burley 21 (nicl / nicl Nic2 / Nic2), Nic2-Burley 21 (Nicl / Nicl) nic2 / nic2) and Nic 1-Nic2-Burley 21 (nicl / nicl nic2 / nic2). Filled bars indicate corresponding mRNA, hatched bars indicate nicotine content.

Figure 6 shows the relative concentration of NtQPT1 mRNA in truncated tobacco plants at various time points compared to untrimmed control plants. Filled bars indicate mRNA content, hatched bars indicate nicotine content.

DETAILED DESCRIPTION OF THE INVENTION

Nicotine is produced in tobacco plants by condensation of nicotinic acid and 4-methylaminobutanal. Figure 1 depicts the nicotine biosynthetic pathway. Two regulatory loci (Nic1 and Nic2) act as co-dominant regulators of nicotine production. Root enzyme assays of single and double mutant Nic genes show that the activities of the two enzymes, quinolate phosphoribosyl transferase (QPRTase) and putrescine methyltransferase, are directly proportional to the level of nicotine biosynthesis. Comparison of the activity of different enzymes with nicotine synthesis activity in tobacco plant tissues (roots and callus) shows that QPRTase activity is strongly correlated with nicotine content (Wagner and Wagner, Planta 165, 532, 1985). Saunders and Bush showed that QPRTase levels in the roots of low nicotine-containing mutants are proportional to nicotine-containing leaves (Plant Physiol 64, 236, 1979).

The present invention encompasses a novel CDNA sequence (SEQ ID NO: 1) encoding plant quinolate phosphoribosyltransferase (QPRTase) sequence SEQ ID NO: 2. Since QPRTase activity is strongly correlated with nicotine content, QPRTase has been reduced in the construction of transgenic tobacco plants (QPRTase). compared to wild-type plants) yields plants with reduced nicotine content in the leaves. The present invention provides a method and nucleic acid constructs for the production of such transgenic plants as well as such transgenic plants. Such methods include expression of antisense NtQPT1 RNA, which lowers QPRTase in tobacco plant roots. Nicotine has also been found in plants of other species and genera, although the amount present is usually much lower than in N. tabacum.

The present invention provides recombinant sense and antisense DNA molecules encoding vectors containing QPRTase or QPRTase antisense RNA molecules and recombinant DNA molecules thereof, as well as transgenic plant cells and plants transformed with such DNA molecules and vectors. The transgenic tobacco plants and cells of this invention are characterized by lower or higher nicotine contents than non-transformed control cells and plants.

Tobacco plants with particularly low nicotine production or no nicotine production are attractive as transgene recipients that express commercially valuable products such as pharmaceuticals, cosmetic components or nutritional supplements. Tobacco is an attractive recipient plant for the transgenes encoding the desired product because tobacco is geethically readily modified geEE 04595 B1 and the biomass produced per acre is very high. Consequently, tobacco plants with reduced nicotine production resources have more resources to produce transgenic products. Methods of transforming tobacco with the transgenes to produce the desired products are known in the art and any suitable method can be used for the low nicotine tobacco plants of the invention.

Tobacco plants of the present invention with reduced QPRTase expression and nicotine content are desirable in the manufacture of low nicotine tobacco products. The tobacco plants of the present invention are suitable for use in any traditional tobacco product, including, but not limited to, pipe, cigar and cigarette tobacco, and chewing tobacco, and can be in any form, including leaf, twist and cut tobacco.

The constructs of the present invention may be useful in the preparation of transgenic plants with increased QPRTase expression and plant nicotine content. Such constructs, methods of using these constructs, and plants produced in this way are desirable in the manufacture of tobacco products having altered nicotine content or in the production of plants whose nicotine content is increased due to insecticidal activity.

The present inventors have found that the TobRD2 gene (Conkling et al., Plant Phys. 93, 1203, 1990) encodes the QPRTase of Nicotiana tabacum and provides herein the NtQPT1 (formerly TobRD2) cDNA sequence and the amino acid sequence of the enzyme to be encoded. Comparison of the amino acid sequence encoded by NtQPT1 with the GeneBank database reveals limited sequence similarity to the bacterial protein encoding quinolate phosphoribosyl transferase (QPRT) (Figure 3).

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Quinolate phosphoribosyl transferase is required for the de novo biosynthesis of nicotine-deninin dinucleotide (NAD) in both prokaryotes and eukaryotes. The tobacco plant has a high content of QPRT in the roots but not in the leaves. In order to determine whether NtQPT1 encodes QPRTase, the inventors used a mutant lacking quinolate phosphoribosyl transferase (NadC ') in Escherichia coli strain TH265. This mutant does not grow on minimal medium lacking nicotinic acid. Expression of the NtQPT1-encoded protein in this bacterial strain results in a NadC? Phenotype (Fig. 4), demonstrating that NtQPT1 encodes QPRTase.

The present inventors investigated the effect of truncation of Nic1 and Nic2 mutants and shoots on steady-state mRNA and nicotine content of tobacco plant NtQPT1. (Clipping off the bud by trimming early flowering is a well-known technique for activating nicotine biosynthesis and transport in tobacco plants and is a common practice in tobacco production.) If NtQPT1 is indeed involved in nicotine biosynthesis, it can be concluded that: 1. NtQPT1 mRNAj2. and 2. NtQPT1 mRNA levels increase after trimming. The NtQPT1 mRNA content of the Nic1 / Nic2 double mutants was found to be approximately 25% of the wild-type mRNA content (Figure 5). NtQPT1 mRNA levels increased approximately eight-fold during the six-hour trimming. This proves that NtQPT1 is a key regulator of the nicotine biosynthetic pathway.

Transgenic plant cells and plants

The regulation of gene expression in the plant cell genome is accomplished by heterologous DNA integration, wherein the transcription of that DNA is controlled by a promoter that is functional in the host cell and wherein the transcribed strand of heterologous DNA is complementary to a DNA strand transcribed from an endogenously regulated gene. The inserted DNA, called antisense DNA, provides an RNA sequence that is complementary to naturally produced (endoEE 04595 B1 gene) mRNA and that inhibits the expression of endogenous mRNA. Such a mechanism for regulating the expression of genes with antisense sequence is not fully known. Unless one theory is to be adhered to, one theory of antisense regulation states that transcription of antisense DNA produces RNA molecules that bind to endogenous mRNA molecules and prevent or inhibit transcription of the latter.

In the methods of the invention, the antisense product may be complementary to the coding or non-coding portion (or both) of the native target RNA. The antisense construct can be introduced into a plant cell by any suitable method and integrated into the plant genome as an inducible or constitutive transcriptionable antisense sequence (Shewmaker et al., U.S. Patents 5,453,566 and 5,107,065, which are incorporated herein by reference in their entirety).

As used herein, exogenous and heterologous DNA (or RNA) refers to DNA (or RNA) that is due to human activity into a cell (or cell precursor). Such heterologous DNA may be a copy of the natural sequence or fragments thereof found in the transformed cell.

To produce a tobacco plant having a higher QPRTase content and thus a lower nicotine content than a non-transformed control plant, the tobacco plant cell may be transformed with an exogenous QPRT antisense transcription unit comprising a portion of the QPRT cDNA sequence, a full length QPRT cDNA sequence with suitable operably linked regulatory sequences. Suitable regulatory sequences include a transcription initiator sequence (promoter) that functions in the plant to be transformed and a polyadenylation / transcription termination sequence. Clones containing the QPRTase sequence operably linked to the antisense orientation and regulatory sequences were identified by standard methods such as restriction analysis, SoutEE 04595 Hern blot hybridization, and nucleotide sequence analysis. The tobacco plants are then successfully regenerated from the transformed cells. Most preferably, the antisense sequence used is complementary to the endogenous sequence. However, slight differences between exo- and endogenous sequences are permissible. It is preferred that the antisense DNA sequence be sufficiently similar to the regulated endogenous sequence in a cell for binding under the harsh conditions described below.

The antisense method is used in many laboratories to produce transgenic plants that have a lower than normal amount of specific enzymes. For example, plants with reduced content of the chalcogen synthase, i.e., the enzyme of the blossom dye biosynthetic pathway, have been obtained by inserting the antisense gene for calcein synthase into the tobacco and petunia genomes. These transgenic tobacco and petunia plants produce lighter colored flowers (Van der Krol et al., 1988, Nature 333, 866869, An Anti-Sense Chalcone Synthase Gene in Transgenic Plant Inhibit Flower Pigmentation). The antisense RNA method has also been successfully used to inhibit the polygalacturonase enzyme in tomatoes (Smith et al., 1988, 334, 724-726, Gene Expression in Transgenic Tomatoes, Nature; Reduction of Polygalacturonase Activity in Tomato Fruit. Natl Acad. Sci USA 85, 8805-8809, 1988), and inhibition of the small ribulose biphosphate carboxylase enzyme in tobacco (Rodermel et al., Nuclear-Organelle Interactions: Nuclear Antisense Gene Inhibits Ribulose Biphosphate Carboxylase Enzyme Levels in Transformed Tobacco Plants, Cell 55, 1988). Alternatively, transgenic plants characterized by higher than normal levels of a given enzyme can be obtained by transforming the plants with the enzyme gene in a sense, i.e., normal orientation. The transgenic tobacco plant of the present invention can be detected by a standard nicotine assay. Transformed plants with reduced QPRTase levels compared to the untransformed control also have lower nicotine levels. TransEE 04595 B1 formulated plants, which have elevated QPRTase levels compared to untransformed control plants, have correspondingly increased nicotine levels.

The heterologous sequence used in the antisense methods of the present invention may be selected to produce an RNA product that is complementary to all or part of the QPRTase mRNA sequence. The sequence may be complementary to any flanking sequence of native mRNA, meaning that it may be complementary to, downstream of, the 5 'end or cap signal sequence of endogenous mRNA sequences, between the cap signal sequence and the initiator codon, and may cover some or all of the non-coding sequence. region, may be linked to a non-coding and coding region, may be complementary to or partially to the coding region, may be complementary to the 3 'end of the coding region, or may be complementary to the untranslated region of the 3' end of the mRNA. Suitable antisense sequences may be at least 13 to 15 nucleotides, at least 16 to 21 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 125 nucleotides, at least 200 nucleotides , or more. The sequences may be truncated or extended at the 3 'or 5' ends.

The specific antisense sequence and its length may vary depending on the degree of inhibition desired, the stability of the antisense sequence, and so on. One skilled in the art will be guided in the selection of the appropriate QPRTase antisense sequence using methods known in the art and the information provided herein. Referring to Figure 2A and SEQ ID NO: 1, the oligonucleotide of the invention may be a continuous fragment of any length of the QPRTase cDNA sequence in an antisense orientation sufficient to produce the desired effect when transformed into a recipient plant cell.

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The present invention can also be used in forward inhibition (i.e., sensory) coin inhibition of nicotine production. The sense DNAs used in the context of the present invention are of sufficient length that, when expressed in a plant cell, inhibit expression of the plant native QPRTase protein as described herein for the plant cell. Such sense DNA may in practice be either genomic or complementary DNA encoding QPRTase, or fragments thereof, which are typically at least 15 nucleotides in length. Methods for determining the length of sense DNA that inhibit expression of a native gene in a cell are known to those skilled in the art.

In an alternative embodiment of the present invention, Nicotiana plant cells are transformed with a DNA construct comprising a DNA segment encoding enzymatic RNA (i.e., ribozyme) molecules targeted to mRNA transcribed from DNA encoding plant QPRTase, i.e., cleaving mRNA. Ribozymes contain a substrate binding domain that binds to the accessible region of the target mRNA and domains that catalyze RNA cleavage, avoiding translation and protein production. The binding domains may comprise antisense sequences complementary to the target mRNA sequence. The catalytic domain may be a hammerhead structure or another such as a hairpin structure. Targets of the ribozyme cleavage site in the RNA sequence can first be identified by scanning the target molecule for ribozyme cleavage sites (i.e., GUA, GUU, or GUC). RNA sequences of short, 15, 20, 30 or more ribonucleotides that correspond to the regions of the target genes containing the cleavage sites can be evaluated for structural properties. The suitability of a potential target can also be evaluated by testing their hybridization ability with complementary oligonucleotides using a ribonuclease protection assay as is known in the art. The DNA encoding the enzymatic RNA molecules can be produced according to known methods (T. Cech et al., U.S. Patent 4,987,071; Keene et al., U.S. Patent 5,559,021; Donson et al., U.S. Patent 5,589,367; Torrence et al., U.S. Patent 5,583,032; Joyce U.S. Patent 5,580,967;

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Gold et al., U.S. Patent 5,595,877; Wagner et al., U.S. Patent 5,591,601; and U.S. Patent 5,622,854, which are incorporated herein by reference in their entirety. Production of such an enzymatic RNA molecule in a plant cell and interruption of the production of QPRTase protein reduces QPRTase activity in the plant cell in the same way as production of an antisense RNA molecule, i.e., interruption of the translation of mRNA required for enzyme production in the cell. The term ribozyme is used herein to describe an RNA-containing nucleic acid that acts like an enzyme (such as an endonuclease) and can be used in the same sense as an enzymatic RNA molecule. The present invention also encompasses ribozyme-encoding DNA introduced into an expression vector, a host cell containing such a vector, and methods for reducing the production of QPRTase in plants by using ribozymes.

The nucleic acid sequences used to carry out the present invention include sequences similar to SEQ ID NO: 1 which encode a protein having quinolate phosphoribosyl transferase activity. This definition includes natural and allelic variants of QPRTase. Thus, a DNA sequence that hybridizes to the DNA sequence of SEQ ID NO: 1 and encodes the expression of QPRTase, particularly plant QPRTase, can also be used to carry out the present invention.

The tobacco QPRT enzyme has many different forms. Many forms of the enzyme may be the result of post-translational modification of a single gene product or many forms of the NtQPT1 gene.

Conditions that allow DNA sequences encoding other proteins having QPRTase activity to be hybridized to the DNA sequence of SEQ ID NO: 1 or other DNA sequences encoding a protein of SEQ ID NO: 2 may be determined by conventional methods. For example, such sequences may be hybridized under less stringent or even stringent conditions (i.e., washing with 0.3 M NaCl, 0.03 M sodium acetate, 0.1% SDS at 60 ° C or even 70 ° C for such DNA). which encodes a protein of SEQ ID NO: 2 and is provided herein as standard conditions for an in situ hybridization assay (J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, 1989, Cold Sprin Harbor Laboratory). In general, such sequences may have 65%, 75%, 80%, 85%, 90% or even 95%, or more, similarity to SEQ ID NO: 1 or DNA sequence encoding the protein of SEQ ID NO: 2 herein. (Maximum similarity between two sequences is compared to determine sequence similarity. To maximize similarity, non-matching segments are allowed in either order. Preferably, these segments have a length of less than 10 nucleotides, preferably less than 5, and more preferably 2 or less.

There are various hybridization methods that allow isolation of cDNA clones with mRNA levels as low as 0.05% of total poly (A).⁺) From RNA (M. Conkling et al., Plant Physiol. 93, 12031211, 1990). Briefly, cDNA libraries are screened using a single-stranded cQNA sample obtained by reverse transcription of plant tissue (i.e., root and / or leaves) mRNA. For differential screening, immerse nitrocellulose or nylon membrane filters in 5xSSC solution, place in a 96-well vacuum collector, transfer 150 μΐ of overnight stationary bacterial culture to each well of the collector, and vacuum allow all liquid to run through a membrane filter. 150 µl of denaturing solution (0.5 M NaOH, 1.5 M NaCl) are pipetted into dry-drawn colonies using a multi-channel pipette and allowed to work for 3 minutes. Use a vacuum as described above, remove the filter from the manifold, neutralize with 0.5 M Tris-HCl (pH 8.0) and 1.5 M NaCl. The filter is heated in a vacuum cabinet for 2 hours and incubated with suitable samples. When using nylon sterilizers and maintaining the motherboard at -70 ° C in 7% DMSO, filters can be repeatedly screened with multiple samples and suitable clones can be obtained after several years of storage.

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As used herein, the term gene refers to a DNA sequence comprising: 1. the upstream (5 ') regulatory sequences, including the promoter, 2. the coding region that defines the gene product, either protein or RNA, 3. the downstream (3') region, contains transcription termination and polyadenylation signals, 4. associated sequences required for efficient and specific expression.

The sequence of the invention may, as an essential part, comprise the sequence set forth herein (SEQ ID NO: 1) or an equivalent nucleotide sequence representing alleles or polymorphic variants of this gene or a coding region thereof.

The term sequence similarity as used in the present specification and claims refers to DNA, RNA or amino acid sequences which have minor and non-essential sequence variations compared to the disclosed and claimed claims and are considered equivalent to the inventive sequences. In this context, minor or insignificant sequence variations mean that similar sequences (i.e., sequences having substantial sequence similarity to DNA, RNA, or protein disclosed and claimed) are functionally equivalent to the disclosed and claimed sequences of the present invention. Functionally equivalent sequences function in substantially the same manner, producing essentially the same nucleic acid compositions as those disclosed herein and claimed.

The DNA sequences provided herein can be transformed into various host cells. Various suitable host cells having desirable growth and handling properties are available in the art.

The terms isolated and substantially pure as used in the present specification and claims for DNA, RNA, polypeptides or proteins means that the DNA, RNA, polypeptides or proteins so described are isolated from their cellular in vivo environment by humans.

Natural DNA sequence or natural DNA sequence herein refers to a DNA sequence that can be isolated from non-transgenic cells or tissue. Natural DNA sequences are those that have not been artificially altered, for example by site-directed mutagenesis. Once the native DNA sequence has been identified, the DNA molecules having the natural sequence may be chemically synthesized or produced by recombinant DNA technology as is known in the art. As used herein, the native plant DNA sequence refers to a sequence that can be isolated from non-transgenic plant cells or tissue. As used herein, the natural tobacco DNA sequence is one that can be isolated from cells or tissue of a non-transgenic tobacco plant.

DNA constructs or transcription cassettes in the context of the present invention include a 5 'to 3' transcriptional promoter described herein, a DNA sequence operably linked to a promoter described herein, and an optional termination sequence including a stop codon recognized by RNA polymerase and a polyadenylase. a recognizable polyadenylation signal. All of these regulatory regions must be able to function in the cells or tissue being transformed. In the embodiment of the present invention, any suitable termination signal may be used, including, but not limited to, a nopaline synthase (nos) terminator, an octapine synthase (ocs) terminator, a CaMV terminator or a native termination signal from the same gene as the transcription initiator region or supra, Rodermel et al., 1988, supra).

As used herein, the term operably linked refers to DNA sequences in one DNA molecule assembled such that the function of one is dependent on the other. Thus, a promoter is operably linked to DNA if it is able to influence the transcription of that DNA transkEE 04595 B1 (i.e., the DNA is under the transcriptional control of that promoter). The promoter is said to be upstream of the DNA, which in turn is downstream of the promoter.

The transcription cassette may be part of a DNA construct that also has at least one replication system. For convenience, a replication system that is effective against Escherichia coli such as ColE1, pSC101, pACYC184 or others is generally used. In this way, the construct obtained at each step after each manipulation can be cloned, sequenced and verified for correctness of manipulation. In addition, E. coli replication system, or in place of the latter, a wide variety of replication systems for different host organisms, such as the P1 incompatible plasmid replication system, i.e., pRK290. In addition to the replication system, the construct often has at least one marker that is active in one or more host organisms, or different markers for different host organisms. That is, one marker may be used for selection in a prokaryotic host and another marker may be used for selection in a eukaryotic organism, particularly a host. Markers can protect against lethal compounds, such as antibiotics, toxins, heavy metals, etc., can provide complementation by transferring prototrophicity to the auxotrophic host or by conferring a visible phenotype on the plant by producing a new compound.

Different fragments containing different constructs, transcription cassettes, markers, etc., can be sequentially inserted by digestion of a suitable replication system with restriction enzymes and insertion of a suitable construct or fragment into a suitable site. After ligation and cloning, the DNA construct can be isolated for further manipulation. All of these methods are sufficiently illustrated in the literature (J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2. 1989, Cold Spring Harbor Laboratory).

Vectors that can be used to transform plant tissue with the nucleic acid constructs of the invention include Agrobacterium vectors and ballistic vectors as well as vectors suitable for DNA-mediated transformation.

The term promoter refers to a region of a DNA sequence that contains the signals necessary for efficient expression of the coding sequence. It may include, but is not limited to, sequences to which RNA polymerase binds, and may also include regions to which other regulatory proteins bind with regions involved in controlling protein translation, and may also include coding sequences.

The promoters used in the present invention may be constitutively active promoters. Many constitutively active promoters that are active in plants are available. For example, the cauliflower mosaic virus (CaMV) 35S promoter, which is constitutively expressed in most plant tissues, is preferred. Alternatively, the promoter may be a sub-specific or a root-specific promoter, as described in more detail below.

The 35S promoter of cauliflower mosaic virus (CaMV) has been used to express antisense sequences in transgenic tobacco plants (Cornelissen et al., Both RNA Levels and Translation Effects are Reduced by Anti-Sense RNA in Nucleic Acids Res. 17, 833-843, 1989; Rezaian et al., Anti-Sense RNAs of Cucumber Mosaic Virus in Transgenic Plants Assessed for Control of the Virus, Plant Molecular Biology 11, 463-471, 1988; Rodermel et al., Nuclear-Organelle Interactions: Nuclear Antisense Gene Inhibits Ribulose Bisphosphate Carboxylase Enzyme Levels in Tranasformed Tobacco Plants, Cell 55, 673681, 1988; Smith et al., 1988, Antisense RNA Inhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes, Nature 334, 724-726; Van der Krol et al., An Anti-Sense Chalcone SynEE 04595 B1 Gene in Transgenic Plant Inhibits Flower Pigmentation, Nature 333, 866-869, 1988).

For expression of QPRTase in the transformed tobacco plant or cells of the invention, the use of the CaMV 35S promoter is preferred. The use of the CaMV promoter to express other recombinant genes in tobacco plant roots has been well described (Lam et al., Site-Specific Mutations Alter In Vitro Factor Binding and Change Promoter Expression Pattern in Transgenic Plants, Proc. Nat. Acad. Sci. USA 86, 7890-7894, 1989; Poulsen et al., Dissection of 5 'Upstream Sequences for Selective Expression of Nicotiana Plumbaginifolia rbcS-8B Gene, Mol. Gen. Genet. 214, 16-23, 1988).

Other promoters that are active only in root tissue (subspecific promoters) are also particularly suitable for the methods of the present invention (Conkling et al., U.S. Patent 5,459,252; Yamamoto et al., The Plant Cell 3, 371, 1991). The root-bark-specific promoter TobRD2 (Conkling et al., U.S. Patent Application SN 08/508,786, now patent; PCT WO 9705261) may be used. All patents cited herein are incorporated by reference in their entirety.

The recombinant QPRTase DNA molecules and vectors used to obtain the transformed tobacco plants and cells of the invention may further comprise a dominant selection marker gene. Suitable dominant selectable markers for use in tobacco plants include, inter alia, antibiotic resistance genes encoding neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT) and chloramphenicol acetyltransferase (CAT). A mutant dihydrofolate reductase gene encoding methotrexate-resistant dihydrofolate reductase is also a dominant selection marker for use in tobacco plants. DNA vectors containing the appropriate antibiotic resistance genes and the corresponding antibiotics are commercially available.

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Transformed tobacco plant cells are selected from the population of surrounding untransformed cells by introducing the mixture of cell populations into a culture medium containing an appropriate amount of antibiotics (or other compounds toxic to tobacco plant cells under normal conditions) to which the dominant selection marker gene confers resistance. Only cells that have been transformed survive and proliferate in such media.

The method of producing the recombinant plants of the present invention generally involves obtaining a plant cell capable of regeneration (typically, such plant cells are located in regenerative tissue). The plant cell is then transformed with a DNA construct containing the transcription cassette of the invention (as described herein) and the transformed plant cell is regenerated with a recombinant plant. Transformation (as described below) is accomplished by methods known in the art, including, but not limited to, bombarding a plant cell with microspheres containing a transcription cassette, infecting the cell with Agrobacterium tumefaciens containing a Ti plasmid carrying the transcription cassette, or any other method. , suitable for the production of transgenic plants.

Many Agrobacterium-based vector systems useful for carrying out the present invention are known. For example, U.S. Patent 4,459,355 discloses a method for transforming common plants, including dicotyledonous plants, with an Agrobacterium strain containing the Ti plasmid. Transformation of forest plants with Agrobacterium-based vector is disclosed in U.S. Patent No. 4,795,855. Schilperoort et al., U.S. Patent No. 4,940,838 discloses a binary Agrobacterium-based vector (i.e., where Agrobacterium contains one plasmid with a vir region but no T region in its Ti plasmid and another plasmid having a T region). but no vir region) which is also useful in practicing the present invention.

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Also, microspheres carrying the DNA construct of the invention and suitable for ballistic transformation of the plant cell are suitable for the preparation of the transformed plant of the invention. The microspheres are introduced into the plant cell to obtain a transformed plant cell and the transformed plant cell is regenerated by the plant. Any suitable ballistic transformation method and apparatus may be used to carry out the invention. Exemplary procedures and apparatus are disclosed by Sanford and Wolf (U.S. Pat. No. 4,945,050) and Christou et al. (U.S. Pat. No. 5,015,580). Using the ballistic transformation method, the transcription cassette may be inserted into a plasmid capable of replication or integration into the cell to be transformed. Such suitable microspheres are, for example, 1 to 5 μτη gold beads. The DNA construct can be applied to the microspheres by any suitable method, for example by precipitation.

The plants may be transformed with the DNA construct of the invention by transforming the plant cell with the protoplast DNA and subsequently regenerating the transformed protoplast plant using methods well known in the art. Fusion of tobacco protoplasts by electroporation with DNA-containing liposomes is known in the art (Shillito et al., 1987, Direct Gene Transfer to Protoplasts of Dicotyledonous and Monocotyledonous Plants, Including Electroporation, Methods in Enzymology 153, 313-336).

As used herein, transformation refers to the introduction of exogenous DNA into a cell to obtain a transgenic cell stably transformed with exogenous DNA.

Transformed cells are induced to regenerate whole tobacco plants using tobacco plant cells and tissue culture methods known in the art. The plant regeneration method is selected to be compatible with the transformation method. The stable presence and orientation of the QPRTase sequence in a transgenic tobacco plant is determined

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QPRTase sequence conformance to Mendelian laws as shown by standard DNA analysis methods used in controlled crossbreeding offspring. After regeneration of the transgenic plant from the transformed cell, the inserted DNA sequence is readily transferable to other tobacco plant varieties by techniques known in the art in everyday plant breeding practice and without undue experimentation.

For example, to investigate transgene divergence, regenerated transformed plants (R_o) to maturity, which is tested for nicotine content and crossbred to produce plants of the R generation. Those plants that carry the transgene are homozygous for the transgene. In order to identify homozygous R 4 plants, R 4 plants are grown to maturity and crossed. Homozygous R 4 plants give R₂- a progeny where each progeny carries a transgene. In the case of heterozygous R 4 offspring, the ratio is 3: 1.

Nicotine acts as a natural pesticide to help protect the tobacco plant from damage caused by pests. Therefore, nicotine-free or low-nicotine plants obtained by the methods of the invention can, if desired, be transformed with a transgene (such as Bacillus thuringiensis) which provides additional insect protection.

Preferred plants for use in the present methods are tobacco or the genus Nicotiana, such as N. tabacum, N. rustica and N. glutinosa. Any strain or variety of tobacco may be used. Strains that already have a low nicotine content, such as the Nic1 / Nic2 double mutants, are preferred.

Any plant tissue capable of subsequent clone propagation by organogenesis or embryogenesis can be transformed with the vector of the invention. The term organogenesis, as used herein, refers to the process by which roots and shoots progressively develop from meristem centers, the term embryogenesis, as used herein, refers to the process by which roots and shoots

EE 04595 ΒΙ develop together in a well-coordinated manner (not sequentially), either from somatic cells or from gametes. The choice of tissue will depend on the clone propagation system available and its suitability for the particular species to be transformed. Examples of suitable tissues are leaf discs, dust blades, embryos, cotyledons, hypocotyls, callus, existing meristem tissues (i.e., apex, leaf bud and root system) and induced meristem tissues (i.e., cotyledon system and hypocotyl meristem).

The plants of the invention may be in various forms. Plants may be transformed and untransformed cell chimeras, clonal transformants (i.e., all cells are transformed and contain a transcription cassette), plants may include grafts of transformed and untransformed tissue (e.g., transformed rootstocks are transplanted onto untransformed citrus grafts). Transformed plants can be propagated in a variety of ways, such as clone propagation or classical breeding methods. For example, plants of the first generation of transformed plants (T,) may be crossed to produce a second generation of homozygous transformed plants (T,).₂) and T₂- Plants are further propagated by classical breeding methods. The transcription cassette may contain a dominant selection marker (nptll) to facilitate breeding.

In the following context, it is clear that plants of the Nicotiana family may be used in the practice of the present invention.

Those familiar with the recombinant DNA techniques described above will appreciate that a full-length QPRTase cDNA molecule or a full-length QPRTase chromosomal gene fused in the sense orientation to suitable, operably linked regulatory sequences may be used to construct transgenic tobacco plants and cells. (One of ordinary skill in the art will recognize that appropriate sense-oriented regulatory sequences for gene expression contain, in addition to the promoter and polyadenylation / transcription termination sequences described above, any known eukaryotic transEE 04595 ΒΙ lation starter sequences.) Such transformed tobacco plants are characterized by higher levels of nicotine content as a non-transformed control herb.

Therefore, it is understood that the use of QPRTase DNA sequences to increase or decrease QPRTase as an enzyme and thus to increase or decrease the nicotine secretion in tobacco plants is within the scope of the present invention.

As used herein, the culture includes a plurality of plants of the invention belonging to the same genus grown together in arable land. Arable land means ordinary land or a greenhouse. Thus, the present invention provides a method for producing plant cultures having altered QPRTase activity and therefore increased or reduced nicotine content compared to non-transformed plant cultures of the same species and variety.

The following examples illustrate the present invention and are not to be construed as limiting the scope thereof.

EXAMPLE 1

Isolation and sequencing

The TobRD2 cDNA (Conkling et al., Plant Phys 93, 1203, 1990) was sequenced and is set forth herein as SEQ ID NO: 1 and the resulting amino acid sequence SEQ ID NO: 2. The deduced amino acid sequence was expected to be a cytosolic protein. Although plant QPTase gene sequences have not been published, comparison of the NtPT1 encoded amino acid sequence with the sequences in the GenBank database (Figure 3) suggests some sequence similarity to common bacterial and other proteins. For example, genes for S. typhimurium, E. coli and N. tabacum are known to encode quinolate phosphoribosyl transferase (QPRTase) activity. The QtTase encoded by NtQPT1 is similar to a derived peptide fragment encoded by the Arabidopsis EST sequence 04595 B1 (expression sequence tag) (Genbank, Accession Number F20096), which may represent part of the Arabidopsis QPTase gene.

EXAMPLE 2

The so-called situ hybridization

To determine the distribution of TobRD2 mRNA transcripts in different root tissues, an in situ hybridization assay was performed on untransformed plants. For in situ hybridization of TobRD2 antisense chain to ToRD2 mRNA in root tissue, the methods described previously (Meyerowitz, Plant Mol. Biol. Rep. 5, 242, 1987; Smith et al., Plant Mol. Biol. Rep. 1987) were used. 5, 237, 1987), Seven-day-old tobacco seedling (Nicotiana tabacum) roots were fixed with glutaraldehyde in phosphate buffer, inserted into Paraplast Plus (Monoject Inc., St. Louis, MO) and cut into 8mm thick sections to obtain both transverse and longitudinal sections. Antisense TobRD2 transcripts were synthesized in vitro³⁵In the presence of S-ATP and used as samples. Labeled RNA was hydrolyzed by alkali treatment to obtain an average of 100-200 nucleotide sequences.

Hybridization was performed in 50% formamide at 42 ° C for 16 hours with labeled RNA (approximately 5 x 10⁶ cpm in 1 ml hybridization solution). After exposure, slides were developed and the preparations were viewed under a conventional dark field microscope.

The hybridization signal was localized in root cortex cells (results not included in patent application). Comparison of images obtained with the same sections under normal and dark field microscopy localized TobRD2 transcripts in root cortex parenchymal cells. No hybridization signal was found in the epidermis and central cylinder.

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EXAMPLE 3

TobRD2 mRNA content in tobacco plant Nic1 and Nic2 mutants and correlation with nicotine content

The steady-state TobRD2 mRNA content was examined in both Nic1 and Nic2 mutant tobacco plants. Nic1 and Nic2 are known to regulate quinolate phosphoribosyl transferase activity and putrescine methyl transferase activity, and are co-dominant regulators of nicotine synthesis. The results shown in Figures 5A and 5B show that Nic1 and Nic2 regulate TobRD2 expression.

RNA was isolated from the roots of wild-type Burley 21 tobacco plants (Niel / Nicl Nic2 / Nic2), Nicl-Burley 21 (nicl / nicl Nic2 / Nic2); Nic2-Burley 21 (Nicl / Nicl nic2 / nic2) and Nicl-Nic2-Burley 21 (nicl / nicl nic2 / nic2).

Four Burley 21 tobacco line (nic) plants obtained from seeds were grown in soil for one month and then placed in a hydroponic chamber for a month in aeration medium. These lines were isogenic except for two lines with a low nicotine content locus and their genotypes were Nicl / Nicl Nic2 / Nic2; Nicl / Nicl nic2 / nic2; nicl / nicl Nic2 / Nic2; nicl / nicl nic2 / nic2, For each genotype, roots were collected from about 20 plants and pooled for RNA isolation. Total RNA (1 μg) isolated from each genotype was electrophoresed through a 1% agarose gel containing 1.1 M formaldehyde and transferred to a nylon membrane (Sambrook et al., 1989). The membranes were hybridized^3ZP is tagged with TobRD2 cDNA fragments. The relative density of TobRD2 transcripts was measured by densitometry. Figure 5 shows the relative transcription levels (filled bars) of all four genotypes relative to Nic / Nic1 Nic2 / Nic2-qa. The relative nicotine content of each of the four genotypes (relative to the Nic1 / Nic1 Nic2 / Nic2 genotype) is indicated by hatched bars.

Figure 5 graphically depicts the relative steady state of TobRD2 mRNA relative to wild-type Burley 21

EE 04595 Bl (Nicl / Nicl Nic2 / Nic2). The TobRD2 mRNA content in the Nic1 / Nic2 double mutants was approximately 25% of the TobRD2 mRNA content of the wild type tobacco plant. Figure 5B compares the relative nicotine content of the nearly isogenic tobacco plant lines studied in this example (filled bars indicate TobRD2 transcript content, hatched bars indicate nicotine content). Nicotine and TobRD2 transcripts were well correlated.

EXAMPLE 4

Effect of truncation on TobRD2 mRNA content

It is well known in the art that removal (trimming) of the tobacco flower head stimulates root growth and increases the nicotine content of the plant leaves. Pruning is a daily practice in industrial tobacco growing, and the optimum time for pruning a particular tobacco plant is readily determined by one of ordinary skill in the art under known growing conditions.

Tobacco plants (N. tabacum SRI) were grown from seed in soil for 1 month and then transferred to sand pots. The plants were grown in a greenhouse for two months until they began to form inflorescences. From four plants, flower heads and shoot tips were trimmed to two knots . After a certain time, some of the roots were removed from each plant and pooled for RNA extraction. Control plants were not truncated. At each time point, total RNA isolated (1 μg) was electrophoresed through a 1% agarose gel containing 1.1 M formaldehyde and transferred to a nylon membrane (Sambrook et al., 1989). The membranes were hybridized³²P is tagged with TobRD2 cDNA fragments. The relative density of TobRD2 transcripts was measured by densitometry. Figure 6 shows the relative content of transcripts at each time point (relative to time zero) in both truncated (filled bars) and untrimmed (hatched bars) plants.

The relative concentration of TobRD2 was determined in root tissue for 24 hours, results are shown in Figure 6 (filled bars show

Transcription levels of TobRD2 in truncated plants, hatched bars in untrimmed plants). TobRD2 mRNA levels in truncated plants increased approximately eight-fold during the six-hour trimming of tobacco plants, whereas no increase was observed in untrimmed control plants during this period.

EXAMPLE 5

Complementation of a QPRTase-lacking bacterial mutant to the DNA sequence of SEQ ID NO: 1

Escherichia coli strain TH265 is a mutant lacking quinolate phosphoribosyl transferase (nadC-) and therefore cannot grow on medium without nicotinic acid.

TH265 cells were transformed with an expression vector (pWS161) containing the DNA sequence of SEQ ID NO: 1, or an empty expression vector (pKK233). The growth of transformed bacteria was compared to that of the TH265 (pKK233) transformants and the untransformed TH265 nadC 'mutant. Growths were compared in minimal medium ME (which did not contain nicotinic acid) and minimal medium ME supplemented with nicotinic acid.

QPTase mutation (nadC) E. coli strain TH265 was obtained from Dr KT Hughes (Hughes et al., J. Bact. 175, 479, 1993). Cells were grown on LB medium and competent cells were prepared as described by Sambrook et al. (1989). The expression plasmid was constructed in pKK2233 (Brosius, 1984) with TobRD2 cDNA, which was cloned under the control of the Tac promoter. The resulting plasmid pWS161 was transformed into TH265 cells. The transformed cells were then seeded on agar plates with minimal medium (Vogel and Bonner, 1956) with or without nicotinic acid (0.0002%). As a control, TH265 cells and pKK2233 transformed TH265 cells were plated on similar plates.

The results are shown in Figure 4. In the medium without nicotinic acid, only TH265 cells transformed with the DNA of SEQ ID NO: 1 grew. These results indicate that the DNA sequence SEQ

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Expression of ID NR: 1 in TH265 bacterial cells gives these cells a NadcS phenotype, confirming that this sequence encodes QPRTase. Therefore, the name TobRD2 was renamed NtQPT1.

EXAMPLE 6

Transformation of tobacco plants

The antisense orientation DNA sequence of SEQ ID NO: 1 was operably linked to plant promoters (CaMV 35S or TobRD2 root bark specific promoter) to produce two different DNA cassettes: CaMV 35S promoter / antisense SEQ ID NO: 1 and TobRD2 promoter / antisense SEQ ID NR: .

Wild type tobacco and low nicotine tobacco line were selected for transformation, i.e. wild type Burley 21 tobacco (Nicl * / Nic2⁺) and homozygous nicl '/ nic2' Burley 21. Both DNA cassettes transformed a number of tobacco plant cells from both lineages. The transformation was carried out using the Agrobacterium vector, i.e. the Agrobacterium binary vector, which contains the Ti-side sequence and the nos promoter (nptll) under the control of the nptll gene (which confers resistance to kanamycin).

Transformed cells were selected and regenerated into transgenic tobacco plants (R_o). R_Q.plants were grown to maturity and tested for nicotine content. The nicotine content of some transformed tobacco plants was significantly lower than that of untransformed control plants.

Then R_gplants and analyzed for transgene divergence in the Rg generation. The R ^ generation was raised to maturity and crossed, the transgene divergence R₂~ generation shows which plants are homozygous for the transgene.

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