Regulation of quinolate phosphoribosyl transferase expression

This invention relates to quinolate phosphoribosyl plant transferase (QPRT-aza) and to the DNA encoding this enzyme. In particular, this invention relates to the use of DNA encoding quinolate phosphoribosyl transferase for the purpose of obtaining transgenic plants having genetically modified nicotine levels, and to the plants thus obtained.

Due to some considerations related to the vicious (addictive) nature of nicotine, the production of tobacco with a low nicotine content is of particular interest. In addition, tobacco plants with extremely low levels of nicotine production, or no nicotine production, are attractive as recipients for transgenes expressing commercially valuable products, such as: pharmaceuticals, cosmetics, or food additives. Various procedures have been devised to remove nicotine from tobacco. However, most of these processes also remove other tobacco ingredients, other than nicotine, thereby affecting tobacco. Tobacco plants with lower nicotine content (approximately 8%) than those found in wild-type tobacco plants were obtained using classical plant regeneration techniques. Tobacco and tobacco plants are desirable with further reductions in nicotine content.

One approach to reducing the level of a biological product is to reduce the amount of an enzyme required in the biosynthesis pathway that leads to that product. If the affected enzyme is naturally found in a limiting amount of velocity (compared to other enzymes required by the biosynthesis pathway), any decrease in the abundance of this enzyme will lead to a decrease in the production of the final product. If the amount of the enzyme is not normally speed-limiting, its presence in a cell must be reduced to speed-limiting levels, in order to decrease pathway efficiency. In contrast, if the naturally occurring amount of enzyme is rate limiting, then any increase in enzyme activity will result in an increase in the end product of the biosynthesis pathway.

In principle, nicotine is formed in the roots of the tobacco plant and is subsequently transported to the leaves, where it is stored (Tso, Physiology and Biochemistry of Tobacco Plants, pp. 233-243, Dowden, Hutchinson & Ross, Stroudsburg, Pa (1972)) . A mandatory step in nicotine biosynthesis is the formation of nicotinic acid from quinolinic acid, a step that is catalyzed by the quinoline phosphorylobyl transferase enzyme (QPRT-aza). QPRT-aza appears to be a rate-limiting enzyme in the nicotinic acid feeding pathway for nicotine synthesis in tobacco. See, for example: Feth et al., "Regulation in Tobacco Callus of Enzyme Activities of the Nicotine Pathway", Planta, 168, pp. 402- 407 (1986); Wagner et al., "The Regulation of Enzyme Activities of the Nicotine Pathway in Tobacco"¹, Physiol. Plant, 68, pp. 667 - 672 (1986). Modification of nicotine content in tobacco plants by antisense regulation of putative methyl transferase expression (PMT-aza) has been proposed in US 5369023 and US5260205 by Nakatani and Malik. Wahad and Malik's PCT application WO 94/28142 describes DNA that encodes PMT and the use of PMT sense and antisense constructs.

The invention relates to an isolated DNA molecule comprising a sequence selected from the sequence group consisting of:

- the sequence SEQ. ID No: 1;

- DNA sequences encoding an enzyme having the amino acid sequence of SEQ. ID No: 2;

- DNA sequences having at least 65% homology to the DNA isolated from points a) and b) and encoding for a quinolate phosphoribosyl transferase enzyme; and

- DNA sequences that differ from the DNA sequences in points a), b) or c) above, due to the degeneration of the genetic code and which encodes for a quinolate phosphoribosyl transferase enzyme.

RO 121968 Β1

The invention further relates to a DNA construct comprising, in the 5-3 'direction, a functional motor in a plant cell and a DNA sequence comprising a coding sequence of the aforementioned quinoliphosphoribosyl transferase, positioned 3 in downstream from the promoter and functionally associated with it.

In another embodiment of the invention, the DNA construct comprises in the 5'-3 'direction a plant 5 promoter and a DNA sequence comprising a coding sequence of the above-mentioned quinolate phosphoribosyl transferase, positioned downstream of the promoter and associated 7. functional with it, the respective DNA sequence being in antisense orientation.

An object of the invention is a plant cell comprising one of the 9 DNA constructs mentioned above.

Another object of the invention is a transgenic plant comprising the cells of 11 mentioned plants.

The invention also relates to a peptide comprising amino acid sequence 13 of the SEQ. ID No .: 2.

The invention also relates to a process for obtaining a transgenic plant cell 15 with decreased expression of quinolate phosphoribosyl transferase comprising:

- providing a plant cell of a known type expressing quinolate 17 phosphoribosyl transferase,

providing an exogenous DNA construct, comprising, in the 5-3 'direction, a promoter 19 functional in a plant cell and a heterologous DNA sequence containing a portion of the sequence encoding quinolate phosphoribosyl transferase mRNA and selected from 21 sequences DNA defined above, the heterologous DNA sequence being functionally associated with the promoter, and 23

- transforming the respective plant cell with said DNA construct, to produce transformed cells, said transformed plant cell having a reduced expression of QPRT -ase compared to an untransformed plant cell, and heterologous DNA comprising at least 25 nucleotides of the sequence of the DNA defined above. 27

Another object of the invention is a transgenic plant of the genus Nicotiana having decreased expression of quinolate phosphoribosyl transferase compared to a non-transfected control plant, comprising transgenic plant cells containing:

- an exogenous DNA construct comprising, in the 5'-3 'direction, a functional promoter in said plant cell 31 and a heterologous DNA sequence comprising a segment of a DNA sequence encoding a quinolate phosphoribosyl transferase mRNA, selected from 33 sequences Mentioned DNA, the respective DNA being functionally associated with the promoter;

- the plant having a reduced expression of quinolate phosphoribosyl transferase 35 compared to an untransformed control plant, and heterologous DNA comprising at least 25 nucleotides of a DNA sequence of the type mentioned. 37

The invention also relates to a descendant of said transgenic plant.

An object of the invention is also a process for reducing the expression of the gene 39 for quinolate phosphoribosyl transferase in a plant cell, comprising:

- growth of a transformed plant cell to contain a heterologous sequence of 41

DNA selected from the aforementioned DNA sequences, wherein a transcribed strand of said heterologous DNA sequence is complementary to endogenous quinol 43 phosphoribosyl transferase mRNA in said cell, wherein transcription of said complementary chain reduces the expression of the quinolate phosphoribosyl transferase gene. 45

The invention also relates to a process for obtaining a tobacco plant with low levels of nicotine in leaves comprising: 47

RO 121968 Β1

- growing a tobacco plant or a descendant thereof, wherein said plant comprises cells containing an exogenous DNA construct, comprising a transcription initiation region, functional in said plant and a heterologous DNA sequence, selected from the DNA sequences according to the invention , functionally linked to the transcription initiation region, wherein the transcribed chain of said heterologous DNA sequence is complementary to endogenous mRNA for quinolate phosphoribosyl transferase in said cells.

The invention also relates to a process for obtaining a transgenic plant cell with increased expression of quinolate phosphoribosyl transferase, which comprises:

- providing a plant cell of a known type to express quinolate phosphoribosyl transferase,

providing an exogenous DNA construct, containing, in the 5'-3 'direction, a functional promoter in a plant cell and a heterologous DNA sequence encoding for quinolate phosphoribosyl transferase and selected from the DNA sequences according to the invention, the DNA sequence heterologous being functionally associated with the promoter;

-transformation of the respective plant cell with said DNA construct to produce a transformed plant cell, the transformed plant cell having an increased expression of quinolate phosphoribosyl transferase compared to an untransformed plant cell.

An object of the invention is also a transgenic plant of the genus Nicotiana having an increased expression of quinolate phosphoribosyl transferase compared to an untransformed control plant comprising transgenic plant cells containing:

- an exogenous DNA construct comprising, in the 5'-3 'direction, a functional promoter in said plant cell and a heterologous DNA sequence encoding a quinolate phosphoribosyl transferase selected from the DNA sequences according to the invention, the heterologous DNA being functionally associated with promoter;

- the plant with an increased expression of quinolate phosphoribosyl transferase and, consequently, increased levels of nicotine in the leaves, compared to an untransformed control plant, as well as its descendants.

The invention also relates to a process for increasing the expression of the quinolate phosphoribosyl transferase gene in a plant cell comprising:

- growing a transformed plant cell to contain an exogenous DNA selected from the DNA sequences according to the invention, wherein the exogenous DNA encodes for quinolate phosphoribosyl transferase.

Another object of the invention is a process for obtaining a tobacco plant with high levels of nicotine in leaves which comprises:

- growing a tobacco plant or a descendant thereof, wherein said plant comprises cells containing an exogenous DNA construct comprising a functional transcription of the initiation region in said plant and a heterologous DNA sequence selected from the DNA sequences according to the invention, linked functional at said transcription initiation region, wherein said heterologous DNA sequence encodes for functionalized quinolate phosphoribosyl transferase in the cells.

The invention also relates to the uses of tobacco plant cells according to the invention, for the manufacture of a tobacco product with a modified nicotine content.

A first aspect of the present invention is an isolated DNA molecule containing: SEQ ID NO: 1; DNA sequences encoding an enzyme having SEQ ID NO: 2; DNA sequences that hybridize with such DNA and encode an enzyme - quinolate phosphoribosyl

RO 121968 Β1 transferase; and DNA sequences that differ from the above DNA due to the degeneracy of the 1 genetic code. A peptide encoded by such DNA is another aspect of this invention. 3

Another aspect of the present invention is a DNA construct containing a promoter operable in a plant cell and a DNA segment encoding an enzyme - quinolate 5 phosphoribosyl transferase, positioned downstream of the promoter and operatively associated with it.

The DNA encoding the enzyme may be in the antisense or sense direction. 7

Another aspect of the present invention is a process for obtaining a transgenic plant cell with reduced expression of quinolate phosphoribosyl transferase (QPRT-aza), 9 by providing a plant cell of a known type with the ability to express quinolate phosphoribosyl transferase; transforming the plant cell with an exogenous DNA construct 11 containing a promoter, and the DNA containing a portion of a sequence encoding the quinolate phosphoribosyl transferase mRNA. 13 Still another aspect of the present invention is a transgenic plant of the Nicotiana species having reduced expression of quinolate phosphoribosyl transferase (QPRT-aza), as compared to an untransformed control plant. The cells of such plants contain a DNA construct that includes a segment of a DNA sequence encoding a mRNA 17 corresponding to plant quinolate phosphoribosyl transferase (QPRT-aza).

Another aspect of the present invention is a process for reducing the expression of a quinolate phosphoribosyl transferase gene in a plant cell, by cultivating a transformed plant cell to contain exogenous DNA, in which it transcribes it. of this exogenous DNA is complementary to the endogenous mRNA in the cell, corresponding to quinoliphosphoribosyl transferase. Complementary chain transcription reduces the expression of the endogenous gene for quinolate phosphoribosyl transferase.

Still another aspect of the present invention is provided by a process for producing a tobacco plant with reduced amounts of nicotine in the leaves of the tobacco plant, by cultivating a tobacco plant with cells containing a sequence of exogenous DNA, wherein a transcribed strand 27 of the exogenous DNA sequence is complementary to the endogenous mRNA corresponding to quinolate phosphoribosyl transferase in cells. 29 Still another aspect of the present invention is a process for obtaining a transgenic plant cell with increased expression of quinoliphosphoribosyl transferase (QPRT-aza), 31 by transforming a plant cell known as producing quinolate phosphoribosyl transferase with an exogenous DNA construct containing a DNA sequence encoding 33 quinolate phosphoribosyl transferase.

Another aspect of the present invention is a transgenic Nicotiana 35 plant, having increased expression (production) of quinolate phosphoribosyl transferase (QPRT-aza), wherein the cells of the transgenic plant contain an exogenous DNA sequence encoding a phosphoribosyl quinolate 37 vegetable transferase.

Another aspect of the present invention is a process for increasing the expression of a quinolate phosphoribosyl transferase gene 39 in a plant cell by cultivating a transformed plant cell to contain exogenous DNA encoding quinolate phosphoribosyl transferase.

Still another aspect of the present invention is a process for producing a tobacco plant 43 having high amounts of nicotine in the leaves, by cultivating a tobacco plant with cells containing an exogenous DNA sequence encoding functional phosphoribosyl quinase transferase 45 in cells. .

Figures. 47

FIG. 1 represents the biosynthesis pathway leading to nicotine. The enzymatic activities known to be regulated by the Nici and Nic2 genes are QPRT-aza (quinolate phosphoribosyl 49 transferase) and PMT-aza (methyl rotase transferase).

RO 121968 Β1

FIG. 2A provides the nucleic acid sequence of the NtQPTI cDNA (SEQ ID NO: 1), together with the coding sequence (SEQ ID NO: 3) represented in capital letters.

- Fig. 2B shows the deduced amino acid sequence (SEQ ID NO: 2) corresponding to the tobacco QPRT -ase, encoded by the NtQPTI cDNA gene.

-fig.3 aligns the deduced amino acid sequence of NtQPT 1 and the similar sequences of Rhodospirillum rubrum, Mycobacterium leprosy, Salmonella typhimurium, Escherichia coli, human, and Saccharomyces cerevisiae.

FIG. 4 shows the results of the complementation of an Escherichia coli mutant lacking quinolate phosphoribosyl transferase (TH265), with the NtQPTI cDNA gene. The cells were transformed with an expression vector carrying the NtQPTI gene; cultivation of TH265 transformed cells expressing the NtQPTI gene, on minimally lacking nicotinic acid medium, demonstrated that the NtQPTI gene encodes QPRT-aza.

- fi g.5 compare the nicotine levels and the relative stationary levels of the NtQPTI gene mRNA in tobacco mutants: wild type Burley 21 (Neither / None Nic2 / Nic2); None - Burley 21 (nic1 / nic 1 Nic2 / Nic2); Nic2 'Burley 21 (Neither / No nic2 / nic2) and Burley 21 NicT and NicZ (nic1 / nic1 nic2 / nic2). Solid bars indicate mRNA transcript levels; Hash bars indicate nicotine levels.

- Fig. 6 graphically represents the relative levels of the mRNA corresponding to the NtQPTI gene, in time, in the tobacco plants superior, compared to the non-performing control plants. Solid bars indicate mRNA transcript levels; The dashed bars indicate nicotine levels.

Nicotine is produced in tobacco plants by condensation of nicotinic acid with 4-methylaminobutanal. The biosynthesis pathway leading to nicotine production is illustrated in FIG. 1. Two regulatory loci (Neither Nic2) act as co-dominant regulators of nicotine production. The enzymatic analyzes of the roots from the mono and double mutants Nic show that the activities of the two enzymes: quinolate phosphoribosyl transferase (QPRT-aza) and methyl transferase rot (PMT-aza), are directly proportional to nicotine biosynthesis levels. A comparison of the enzymatic activity of tobacco plant tissues (root and callus) with different nicotine synthesis capacities shows that QPRT-activity is closely correlated with nicotine content (Wagner and Wagner, Plant 165: 532 (1985)). Saunders and Bush (Plant Physiol. 64: 236 (1979)) showed that the QPRT-level of the roots of the low-nicotine mutant roots is proportional to the nicotine levels of the leaves.

The present invention comprises a novel cDNA sequence (SEQ ID NO: 1) encoding a plant quinolate phosphoribosyl transferase (QPRT-azase) having SEQ ID NO: 2. As the activity of QPRT-aza is closely correlated with the nicotine content, the construction of transgenic tobacco plants in which the levels of QPRT-aza are low in the roots of the plant (compared with the levels of the wild type plants) leads to plants with low levels of nicotine in leaves. The present invention provides nucleic acid methods and constructs for the production of such transgenic plants, as well as such transgenic plants. Such methods include expression of antisense RNA for the NtQPTI gene, thereby reducing the amount of QPRT -ase from tobacco roots. Nicotine has also been found in other plant species and families than tobacco, although the quantity present is usually much lower than in Nicotiana tabacum.

The present invention also provides recombinant sense and antisense DNA molecules encoding QPRT-aase or QPRT-RNA corresponding antisense molecules, and vectors containing those recombinant DNA molecules; as well as transgenic plant cells and plants transformed with those DNA molecules and vectors. The transgenic tobacco cells and plants of the present invention are characterized by a lower or higher nicotine content compared to the non-transformed control cells and tobacco plants.

RO 121968 Β1

Tobacco plants with extremely low levels of nicotine production, or without 1 nicotine production, are attractive as recipients for transgenes expressing commercially valuable products such as: pharmaceuticals, 3 cosmetic components or food additives. Tobacco is attractive as a host plant for a transgene that encodes a desired product because it is easy to manipulate through genetic engineering and produces a very large amount of biomass per acre; tobacco plants with low resources for nicotine production will therefore have additional resources available 7 for the production of transgenic products. Methods of transforming tobacco with transgenes producing desired products are known in the art; any suitable technique 9 can be used in the case of tobacco plants having a low nicotine content from this invention. 11

Tobacco plants according to the present invention, with a reduced expression of QPRT -ase and low nicotine levels, will be desirable in obtaining tobacco products having a low nicotine content. Tobacco plants according to the present invention will be suitable for use in any traditional tobacco product, including, but not limited to, pipe, 15 cigarette or cigarette tobacco and chewing tobacco, but may be in any form, including tobacco leaves. , chopped tobacco or cut tobacco. 17

The constructs of the present invention may also be useful in providing transgenic plants with increased expression of QPRT -ase and a high nicotine content in 19 plants. Such constructs, methods using these constructs, as well as plants obtained in this way may be desirable in the production of tobacco products having an altered nicotine content, or in the production of plants with a high nicotine content, used as a result. nicotine insecticides. 2. 3

The present inventors have found that the TobRD2 gene (see Conkling et al.,

Plant Phys. 93, 1203 (1990)) encode a QPRT -ase from Nicotiana tabacum and provided 25 cDNA sequences of NtQPTI (formally called TobRD2), as well as the amino acid sequence of the encoded enzyme. Comparisons of the amino acid sequence corresponding to the NtQPTI 27 gene with the databases in the Gene Bank reveal a limited sequence similarity with the bacterial proteins encoding quinolate phosphorylase transferase (QPRT-aza) (Figure 3). 29

Chinolate phosphorylase transferase is required for de novo biosynthesis of nicotinadenindinucleotide (NAD) in both prokaryotes and eukaryotes. In the case of tobacco, 31 large quantities of QPRT -ase were detected in the roots, but not in the leaves. To determine whether the NtQPTI gene encodes QPRT-aza, the present inventors used the bacterial strain of 33 Escherichia coli (TH265) - a mutant deficient in quinolate phosphorylase transferase (nadC).⁺). This mutant cannot grow on a minimal medium without nicotinic acid. However, expression of NtQPT 1 protein in this bacterial strain conferred the NadC * phenotype (Figure 4), confirming that NtQPTI encodes QPRT-aza. 37

The present inventors examined the effects of the Ni and Nic2 mutants in tobacco, as well as the effects of cutting the tobacco plant's upper part on the stationary levels of the 39 mRNA corresponding to the NtQPTI gene and on nicotine levels. (It is well-known that removing apical dominance by cutting the upper part in the flowering stage results in increased levels of nicotine biosynthesis and transport in tobacco, and it is a standard practice in tobacco production). If the NtQPTI gene is indeed 43 involved in nicotine biosynthesis, then it is expected that: (1) the levels of mRNA corresponding to the NtQPTI gene are lower in the Nic1INic2 double mutants and (2) levels 45 of the mRNA corresponding to the NtQPTI gene to either grown after the upper part is cut.

It was found that the mRNA levels corresponding to the NtQPTI gene In the case of double mutants 47 Nic1 / Nic2 are approximately 25% of those of the wild type (Fig. 5). Moreover, after 6 h

Since the cutting of the upper part, the levels of mRNA corresponding to the NtQPTI gene in tobacco plants have increased approximately 8-fold. Therefore, it has been established that the NtQPT 1 gene is a key regulatory gene in the nicotine biosynthesis pathway.

Transgenic plant cells and plants

Regulation of gene expression in plant cell genomes can be accomplished by integrating heterologous DNA under the transcriptional control of a promoter that is functional in the host, and in which, the transcribed chain of heterologous DNA is complementary to the DNA chain that is transcribed from the endogenous gene. which needs to be adjusted. Introduced DNA, in the form of antisense DNA, provides an RNA sequence that is complementary to naturally occurring (endogenous) mRNAs and inhibits the expression of endogenous mRNA. The mechanism of such regulation of antisense gene expression is not fully understood. While not intended to be limited to a single theory, it has been noted that a theory of antisense regulatory proposals for that transcription of antisense DNA produces RNA molecules that bind / inhibit or inhibit the transcription of endogenous mRNA molecules.

In the methods of the present invention, the antisense product may be complementary to the coding or non-coding portions (or both) of naturally occurring target RNA. The antisense construct can be introduced into plant cells by any suitable means and can be integrated into the plant genome for inducible or constitutive transcription of the antisense sequence. See, for example, patents US5453566 and US5107065 by Shewmaker et al.

As used herein, the names of heterologous DNA (or RNA) refer to the DNA (or RNA) that has been introduced into a cell (or cell ancestor) through human efforts. Such heterologous DNA may be a copy of a sequence that is naturally found in the cell thus transformed, or fragments thereof.

In order to produce a tobacco plant with low levels of QPRT -ase and, therefore, with a lower nicotine content than an untransformed tobacco control plant, a tobacco cell can be transformed with an exogenous, antisense transcriptional unit for QPRT. containing: a partial cDNA sequence for QPRT; a whole sequence from the cDNA corresponding to the QPRT; a partial chromosomal sequence for QPRT; or a whole chromosome sequence for QPRT in antisense orientation with the appropriate regulatory sequences operationally linked. Suitable regulatory sequences include a transcription initiating sequence (promoter) operable in the plant undergoing transformation and a polyadenylation / transcription termination sequence. Next, standard techniques such as restriction mapping, Southern blot hybridization, and nucleotide sequence analysis were used to identify carrier clones of QPRT-sequence sequences in antisense orientation, operatively linked to regulatory sequences. Tobacco plants are then regenerated from successfully transformed cells. Most convenient is that the antisense sequence used is complementary to the endogenous sequence; however, minor variations in exogenous and endogenous sequences can be tolerated. It is preferable that the DNA antisense sequence has sufficient sequence similarity to be able to bind to the endogenous sequence in the cell to be regulated, under strict conditions, as described below.

In different laboratories, antisense technology has been used to create transgenic plants characterized by smaller than normal quantities of some specific enzymes. For example, plants with low levels of calcon synthase - an enzyme from a biosynthesis pathway of a floral pigment - were obtained by inserting an antisense gene corresponding to calcon synthase in the genome of tobacco and petunia plants. These transgenic tobacco and petunia plants produce flowers with a lower color than normal (Van der Krol et al., An Anti-Sense Chalcone Synthase Gene in Transgenic Plants Inhibits Flower Pigmentation,

RO 121968 Β1

Nature, 333, pp. 866 - 869 (1988)). Also, antisense RNA technology has been successfully used to inhibit polygalacturonase enzyme production in tomatoes (Smith et al., Antisense RNA Inhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes, 3 Nature, 334, pp. 724-726 (1988); Sheehy et al., Reduction of Polygalacturonase Activity in Tomato Fruit by Antisense RNA, Proc Natl. Acad. Sci. USA, 85, pp. 8805 -8809 (1988), 5 and the small subunit of the tobacco ribulose bisphosphate carboxylase enzyme (Rodermel et al., Nuclear-Organelle Interactions: Nuclear Antisense Gene Inhibits Ribulose Bisphosphate 7 Carboxylase Enzyme Levels in Transformed Tobacco Plants, Cell, 55, pp , 673 - 681 (1988)).

Alternatively, transgenic plants characterized by quantities greater than 9 normal ones in a given enzyme can be created by transforming the plants using the gene for that enzyme in a sense orientation (ie normal). The nicotine levels of the 11 transgenic tobacco plants of the present invention can be detected by standard nicotine determinations. Transformed plants in which the level of QPRT-azae is low, compared to that of the control plants 13 not transformed, will therefore have a reduced nicotine level, compared to the control;

transformed plants in which the level of QPRT-azase is increased compared to the control plants not transformed will consequently have an increased nicotine level compared to the control.

The heterologous sequence used in the antisense methods of the present invention may be selected so as to result in obtaining an RNA product complementary to the entire mRNA sequence for QPRT -ase, or to a portion thereof. The sequence 19 may be complementary to any neighboring sequence of the natural mRNA, that is, it may be complementary to the endogenous mRNA sequence adjacent to the 5 'end or end 21 which is isolated during the mRNA maturation process, downstream from the latter end. mentioned, or between this end and the initiating codon and may cover the whole / or only a portion of the non-coding region, may unite, by a bridge, the non-coding region with the coding region, it may be complementary to all / or part of the coding region, complementary to the 3 'terminal end of the coding region, or complementary to the 3' - untranslated region of the mRNA. Suitable antisense sequences may consist of at least 27 to 13 nucleotides, at least 16 to 21 nucleotides, at least 20 nucleotides, at least about 30 nucleotides, at least about 50 nucleotides, at least 29 at least 75 nucleotides. nucleotides, at least 100 nucleotides, at least 125 nucleotides, at least 150 nucleotides, at least 200 nucleotides, or more. In addition, the sequences may be extended 31 or shortened at their 3 'or 5' ends.

The particular antisense sequence and the length of the antisense sequence will vary depending on the desired degree of inhibition, the stability of the antisense sequence and other similar criteria.

Someone skilled in the art will be guided in the selection of appropriate 35 QPRT-antisense sequences, using the techniques available in the art and the information provided herein. With reference to Figure 2A and SEQ ID NO: 1 of the present invention, an 37 oligonucleotide of the present invention may be a continuous fragment of the QPRT-sequence of cDNA in antisense orientation, of any length sufficient to effect the effects. desired to be transformed into a host plant cell.

The present invention may also be used in the co-suppression methods of nicotine production. The sense DNAs used in carrying out the present invention are of sufficient length to suppress (when expressed in a plant cell) native expression of the QPRT-aza plant protein, as described herein, in that plant cell. Such sense DNAs can be, in essence, whole genomic or complementary DNA encoding 45 QPRT enzymes, or a fragment thereof, with such fragments typical of at least

RO 121968 Β1 nucleotides, in length. At the disposal of experts in the field there are methods of determining the length of sense DNA that result in suppressing the expression of a native gene in a cell.

In an alternative embodiment of the present invention, Nicotiana plant cells are transformed by means of a DNA construct containing a DNA segment encoding an enzyme RNA molecule (ie, a ribozyme), an enzyme RNA molecule that is directed to (ie cleaves) mRNA transcript of DNA encoding plant QPRT, as described herein. Ribozymes contain substrate binding domains that bind to accessible regions of the target mRNA, as well as domains that catalyze RNA cleavage, avoiding protein translation and production. The binding domains may contain antisense sequences complementary to the target mRNA sequence; the catalytic motif (drawing) may be a hammerhead motif or other reasons, such as the staple motif. The cleavage sites of an RNA target can first be identified by scanning the target molecule from the point of view of the cleavage sites of the ribozyme (eg GUA, GUU or GUC sequences). Once identified, short RNA sequences of 15, 20, 30 or more ribonucleotides, corresponding to the region of the target gene containing the cleavage site, can be evaluated for the purpose of providing structural features. With the help of ribonuclear protection tests known in the art, the suitability (similarity) of the candidate targets can be evaluated by testing their availability for hybridization with complementary oligonucleotides. DNA encoding enzyme RNA molecules can be produced according to known techniques. See, for example, T. Cech et al., US 4987071; Keene et al., US 5559021; Donson et al., US 5589367; Torrence et al., US 5583032; Joyce, US 5580967; Gold et al., US 5595877; Wagner et al., US 5591601; and US 5622854. The production of such an enzymatic RNA molecule in a plant cell and the disruption of QPRT -ase protein production lead to reduced QPRT -ase activity in plant cells in the same way (in essence) as the production of an antisense RNA molecule: that is, by interrupting translation. MRNA in the cell that produces the enzyme. The term ribozyme is used here to describe a nucleic acid containing RNA that functions as an enzyme (such as an endoribonuclease) and may be used instead of the term enzyme RNA molecule. The present invention also includes: DNA encoding ribozymes; DNA encoding ribozymes that have been inserted into an expression vector; host cells containing such vectors, as well as methods using ribozymes, to reduce QPRT -ase production in plants.

The nucleic acid sequences used in carrying out the present invention include those having a sequence similarity to SEQ ID NO: 1 and encoding a protein having quinolate phosphoribosyl transferase activity. This definition is intended to include natural allelic variations in QPRT-azic proteins. Thus, DNA sequences that hybridize with DNA having the sequence SEQ ID NO: 1 and encode the expression of QPRT-aza, in particular plant QPRT-azic enzymes, may also be used in carrying out the present invention.

There may be multiple forms of the QPRT-enzyme in tobacco. These multiple forms of an enzyme may be due to post-translational modification of a single gene product, or multiple forms of the NtQPTI gene.

The conditions that allow other DNA sequences encoding the expression of a protein having QPRT-azic activity to hybridize with DNA having the sequence SEQ ID NO: 1 or with other DNA sequences encoding the protein represented by SEQ ID NO: 2, can be determined by -a routine way. For example, hybridization of such sequences may be carried out under conditions of reduced rigor, or even under rigorous conditions (e.g. conditions

RO 121968 Β1 represented by rigorous washing with 0.3 M NaCl, 0.03 M sodium citrate, 0.1% SDS, at 1 60 ° C or even 70 ° C for the DNA encoding the protein represented herein by SEQ ID NO: 2, in a standard in situ hybridization test. See J. Sambrook et al., 3 Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory). In general, such sequences will be at least 65% similar, 75% similar, 80% similar, 85% 5 similar, 90% similar, or even 95% similar, or even more, with the sequence represented here by SEQ ID NO: 1, or with DNA sequences encoding proteins having 7 SEQ ID NOs: 2. (Sequence similarity determinations were made with the two sequences aligned for maximum matching; to increase the matching degree, 9 breaches are allowed in either of the two similar sequences. Lengths of the 10 or less, more preferred breaches are preferred. are the lengths of the breaches of 5 or less, and 11 the lengths of the breaches of 2 or less are even more convenient).

There are differential hybridization procedures that allow isolation of cDNA clones of 13 whose mRNA levels are approximately 0.05% poly (A⁺) RNA. See M. Conkling et al., Plant Physiol. 93, 1203 - 1211 (1990). Briefly, cDNA banks are screened, using single-stranded cDNA probes, from the point of view of reverse transcribed mRNA from plant tissue (eg, roots and / or leaves). For differential screening 17 proceed as follows: a nitrocellulose or nylon membrane is soaked 5 times in SSC, placed in a vacuum manifold having 96 wells, 150 µL of stationary culture 19 overnight are transferred from a stock plate (mother) in each well and subjected to vacuum pressure until all liquid passes through the filter. 150 pl_ of the denaturing solution (0.5 M NaOH, 21 1.5 M NaCl) are placed in each well with a pipette and allowed to stand for approximately 3 min. It is aspirated as above and the filter is removed and neutralized in 0.5 M Tris-HCl 23 (pH 8.0), NaCl 11.5 M. The filter is then dried in vacuo for 2 h and incubated with the relevant probes. By using nylon membrane filters and keeping the main plates stored at -70 ° C 25 in 7% DMSO, the filters can be screened multiple times, with multiple probes, and the particular clones can be recovered after several years of storage.27

As used herein, the term "gene" refers to a DNA sequence that incorporates: (1) regulatory signals, including the promoter, located upstream of the 5 'end, 29 (2) a coding region specifying the product (protein) or RNA) of the gene, (3) regions downstream of the 3 'end, including polyadenylation and transcription termination signals31 and (4) associated sequences required for efficient and specific expression.

The DNA sequence according to the present invention may consist essentially of the 33 sequence provided herein (SEQ ID NO: 1), or of equivalent nucleotide sequences representing allelic or polymorphic variants of these genes, or of coding regions 35 thereof. .

The use of the phrase "substantial sequence similarity" in the present specification and in 37 claims refers to the fact that DNA, RNA or amino acid sequences that have slight variations and have no consequences from the actual sequences described and claimed in the present application. are considered to be equivalent to the sequences of the present invention. From this point of view, "slight and non-consequence sequential variations" means that "similar" sequences 41 (ie, sequences that have a substantial similarity of sequences to the DNA, RNA or proteins described and claimed in the present application ) will be functionally equivalent to the sequences described and claimed in the present invention. Functionally equivalent sequences will essentially function in the same manner to produce, in the main, the same compositions as the nucleic and amino acid compositions described and claimed in the present work. 47

RO 121968 Β1

The DNA sequences provided in the present invention can be transformed into a variety of host cells. In the field there are and are easily accessible a variety of suitable host cells, having the desired cultivation and handling properties.

The use of the expression "isolated" or "substantially pure" herein and in the claims as a modifier of DNA, RNA, polypeptides or proteins means that the DNA, RNA, polypeptides or proteins so designated have been separated from their cellular media in alive, through the efforts of the people. As used herein, a "native DNA sequence" or "natural DNA sequence" means a DNA sequence that can be isolated from non-transgenic tissues or cells. Native DNA sequences are those that have not been artificially altered, such as mutagenesis directed to a particular site. Once the native DNA sequences are identified, the DNA molecules having native DNA sequences can be chemically synthesized or produced using recombinant DNA processes, as is known in the art. As used in the present invention, a native plant DNA sequence is one that can be isolated from non-transgenic plant tissues or cells. As used in the present invention, a native DNA sequence of tobacco is that which can be isolated from non-transgenic tobacco tissues or cells.

The DNA constructs, or "transcription cassettes" of the present invention include, from the 5 'end to the 3' end in the direction of transcription: a promoter, as previously discussed, a DNA sequence, as previously discussed in the specification, operationally associated with the promoter; and optionally, a termination sequence that includes the stop signal for RNA polymerase and a polyadenylation signal for polyadenylase. All these regulatory regions must be able to operate in the cells of the tissue to be transformed. Any suitable termination signal can be used in carrying out the present invention, such examples including but not limited to: nopalin synthase terminator (nos), octapin synthase terminator (ocs), CaMV terminator or native termination signals derived from the same gene as the region of transcriptional initiation or derived from a different gene. See, for example: Rezian et al. (1988) above, and Rodermel et al. (1988), above.

The term "operatively associated", as used herein, refers to DNA sequences on a single DNA molecule, which are associated in such a way that the functioning of one is impaired by the other. Thus, a promoter is operatively associated with a DNA when it is capable of affecting the transcription of that DNA (ie, the DNA is under the transcriptional control of the promoter). It is said that the promoter is upstream of DNA, which is said to be "downstream" from the promoter.

The transcription cassette can be provided in a DNA construct that also has at least one replication system. For convenience, a functional replication system in Escherichia coli, such as ColE1, pSC101, pACYC184, or the like, is used. In this way, at each stage, after each manipulation, the resulting construct can be donated, sequenced and the correctness of the manipulation determined. In addition, or in place of the E.coli replication system, a wide range of host systems for replication, such as the replication system of the incompatibility P-1 plasmid, for example, pRK290, may be used. Outside the replication system, there is frequently at least one marker present, which may be useful in one or more hosts, or different markers for different hosts. That is, a marker may be used for selection in a prokaryotic host, while another marker may be used for selection in a eukaryotic host, in particular the plant host. Markers can provide protection against a biocide, such as antibiotics, toxins, heavy metals, or others of this type; they can offer complementarity through

RO 121968 Β1 sharing the prototrophy with an auxotrophic host; or they can provide a visible phenotype by producing 1 of a new compound in the plant.

Different fragments containing various constructs, transcription cassettes, markers and the like 3 can be introduced consecutively, by cleaving the restriction enzyme of a suitable replication system and inserting the particular construct or fragment into the available site. 5

After ligation and donation, the DNA construct can be isolated for further manipulation. All these techniques are widely exemplified in the literature, as J. Sambrook et al., 7

Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory).

The vectors provided in the present invention that can be used to transform plant tissue with nucleic acid constructs include both Agrobacterium vectors and ballistic vectors, as well as vectors suitable for DNA-mediated transformation. 11

The term "promoter" refers to a region of a DNA sequence that incorporates the signals required for efficient expression of the coding sequence. 13 It includes sequences to which an RNA polymerase binds, but is not limited to such sequences and may include regions to which other regulatory proteins bind, together with regions involved in the control of protein translation, as well as coding sequences.

The promoters used in carrying out the present invention may be constitutively active promoters. 17 Numerous constituent active promoters that are operable in plants are accessible. A convenient example is the 35S promoter of the cauliflower mosaic virus (CaMV = Cauliflower 19 Mosaic Virus) which is constitutively expressed in most plant tissues. Alternatively, the promoter may be a root-specific promoter, or root-specific cortex 21 promoter, as explained in more detail below.

Antisense sequences were expressed in transgenic tobacco plants, using 23 cauliflower mosaic virus (CaMV) 35S promoter. See, for example, Cornelissen et al., "Both RNA Level and Translation Efficiency are Reduced by 25 Anti-Sense RNA in Transgenic Tobacco," Nucleic Acids Res. 17, pp. 833-843 (1989); Rezaian et al., “Anti-sense RNAs of Cucumber Mosaic Virus in Transgenic Plants 27 Assessed for Control of the Virus, Plant Molecular Biology 11, pp. 463 - 471 (1988); Rodermel et al., "Nuclear-Organelle Interactions: Nuclear Antisense Gene Inhibits 29 Ribulose Bisphosphate Carboxylase Enzyme Levels in Transformed Tobacco Plants", Cell 55, pp. 673 - 681 (1988); Smith et al., "Antisense RNA Inhibition of Polygalacturonase 31 Gene Expression in Transgenic Tomatoes", Nature 334, pp. 724-726 (1988); Van der Krol et al., An Anti-Sense Chalcone Synthase Gene in Transgenic Plants Inhibits Flower 33 Pigmentation ”, Nature 333, pp. 866 - 869 (1988).

It is preferred to use the 35S CaMV promoter for expressing QPRT -ase in the transformed tobacco plants and cells of this invention. The use of the CaMV promoter for the expression of other recombinant genes in tobacco roots has been well described (Lam et al. 37, "Site-Specific Mutations Alter In Vitro Factor Binding and Change Promoter Expression Pattem in Transgenic Plants", Proc. Nat. Acad. Sci USA 86, pp. 7890 - 7894 39 (1989); Poulsen et al., "Dissection of 5 'Upstream Sequences for Selective Expression of the Nicotiana plumbaginifolia rbcS-8B Gene", Mol. Gender. Genet. 214, pp. 16-23 (1988)). 41

Also, other promoters that are active only in root tissues (root specific promoters) are particularly suited to the methods of the present invention. See, for example, US Patent 5,459,252 to Conkling et al. Yamamoto et al., The Plani Cell, 3: 371 (1991). The promoter specific to the root cortex - TobRD2 may also be used. See, for example, US patent application SN 08 / 508,786, admitted to Conkling et al; PCT WO 9705261. All of the patents cited herein will be incorporated herein by reference in their entirety.

RO 121968 Β1

The recombinant QPRT-DNA molecule molecules and the vectors used to produce the transformed tobacco cells and plants of the present invention may additionally contain a dominant selectable marker gene. Dominant selectable markers suitable for tobacco use include, among others, antibiotic resistance genes encoding neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT) and chloramphenicol acetyltransferase (CAT). Another well-known dominant selectable marker, suitable for tobacco use, is a mutant organ for dihydrofolate reductase, which encodes methotrexate-resistant dihydrofolate reductase. DNA vectors containing genes suitable for antibiotic resistance as well as the appropriate antibiotics are commercially available.

Transformed tobacco cells are separated from the surrounding population of untransformed cells by placing the mixed population of cells in a culture medium containing an appropriate concentration of the antibiotic (or other tobacco cell toxic compound) to which the dominant selectable marker gene product confers. resistance. Thus, only those tobacco cells that have been transformed will survive and multiply.

Methods of obtaining the recombinant plants of the present invention generally involve, firstly, the supply of a plant cell capable of regeneration (the plant cell being typically in a tissue capable of regeneration). The plant cell is then transformed with a DNA construct containing a transcription cassette of the present invention (as described above) and a recombinant plant is regenerated from the transformed plant cell. As explained below, the transformation step was performed by techniques that are known in the art including, but not limited to: bombardment of the plant cell with microparticles carrying the transcription cassette; infecting the cell with an Agrobacterium tumefaciens containing a Ti plasmid carrying the transcription cassette; or any other suitable technique for producing a transgenic plant.

Numerous Agrobacterium vector systems are known, useful in carrying out the present invention. For example, US Patent 4,459,355 discloses a method for transforming susceptible plants, including dicotyledonates, with an Agrobacterium strain containing Ti plasmid. The transformation of woody plants with an Agrobacterium vector is presented in US Patent 4,955,855. In addition, US Patent 4940838 to Schilperoort et al. discloses a binary vector of Agrobacterium (i.e., a vector in which Agrobacterium contains a plasmid having the vir region of a Ti plasmid, but not the T region, and a second plasmid having a T region, but not the vir region) useful in carrying out the present invention .

The microparticles carrying a DNA construct of the present invention - microparticles that are suitable for the ballistic transformation of a plant cell are also useful for obtaining the transformed plants of the present invention. The microparticle is propelled into a plant cell to produce a transformed plant cell from which a plant regenerates. For the practical implementation of the present invention any methodologies and any ballistic transformation apparatus of the cell can be used. Exemplary apparatus and procedures have been described by Sanford and Wolf in US Patent 4945050 and Christou et al. In US Patent 5015580. When using ballistic transformation processes, the transcription cassette can be incorporated into a plasmid capable of replication in / or integration into the cell to be transformed. Examples of microparticles suitable for use in such systems include 1-5 pm gold spheres. The DNA construct can be deposited on the microparticle by any suitable technique, such as precipitation.

Plant species can be transformed with the DNA construct of the present invention by DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from transformed protoplasts, in accordance with procedures well known in

RO 121968 Β1 domain. The fusion of tobacco protoplasts with liposomes containing 1 is known in the art

DNA or via electroporation (Shillito et al., "Direct Gene Transfer to Protoplasts of Dicotyledonous and Monocotyledonous Plants by a Number of Methods, Including 3 Electroporation", Methods in Enzymology 153, pp. 313 - 336 (1987)).

As used herein, the transformation refers to the introduction of exogenous DNA into cells to produce transgenic cells stably transformed with exogenous DNA. 7

Transformed cells are induced to regenerate intact tobacco plants by applying cell culture techniques and tobacco tissues - techniques that are well known in the art. The method of plant regeneration is chosen in such a way that it is compatible with the method of transformation. The stable presence and orientation of the QPRT-aza sequence in the 11 transgenic tobacco plants can be verified by the Mendelian inheritance of the QPRT-aza sequence, as revealed by the standard DNA analysis methods applied to the resulting offspring 13 from controlled crosses. After the regeneration of transgenic tobacco plants from transformed cells, the introduced DNA sequence is rapidly transferred to other tobacco varieties through 15 conventional plant breeding practices and without excessive experimentation.

For example, to analyze transgene segregation, transformed plants are regenerated (R_a) can be grown to maturity, tested from the nicotine content point of view and redone to produce plants R ,. A percentage of R. plants bearing 19 transgenes are homozygous for the transgene. For the identification of homozygous plants R_1fthe transgenic plants Ri are grown to maturity and restored. Rt 21 homozygous plants will produce the R offspring₂ where each downstream plant carries the transgene; descendant of heterozygous plants R₁ segregates 3: 1. 2. 3

Because nicotine serves as a natural pesticide, it helps protect tobacco plants from the onslaught of pests. Therefore, it may be desirable to further transform low- and / or nicotine-free plants produced by the present methods with the aid of a transgene (such as Bacillus thuringiensis) that will provide additional insect protection 27.

A preferred plant for use in these methods is the Nicotiana species, or tobacco, including: N. tabacum, N. rustica and N. glutinosa. Any tobacco strain or variety can be used. Preferred are strains that already have a low nicotine content, such as 31 Nic1 / Nic2 double mutants.

Any plant tissue capable of subsequent clonal propagation, either by organogenesis, 33 or by embryogenesis, can be transformed with a vector of the present invention. The term "organogenesis", as used by the present paper, designates a process through 35 which, from meristematic centers, develops sequentially (in stages) stems and roots; the term embryogenesis, as used herein, means a process by which the 37 stems and roots develop together, in concert (not sequentially), either from somatic cells or from gills. The particular tissue chosen will vary depending on the clonal propagation systems 39 available to / and most suitable for the particular species that are subject to transformation. Exemplary target tissues include: leaf disks; tissue from: pollen, embryo, 41 cotyledons, hypocotyls, calus; existing meristematic tissue (eg apical meristems, axillary buds and root meristems); as well as induced meristemic tissue (eg, cotyledon meristem 43 and hypocotyl meristem).

The plants of the present invention can take a variety of forms. Plants can be chimeras of 45 transformed cells and untransformed cells; the plants can be clonal transformants (eg all cells transformed in the sense to contain the transcription box); the plants may contain grains 47 transformed or untransformed tissues (eg, a trunk of grafted transformed root

RO 121968 Β1 on an unprocessed seedling, for citrus species). Transformed plants can be propagated through a variety of ways, such as using clonal propagation or classical breeding techniques. For example, the first generation (or T1) of transformed plants can be regenerated to give the second homozygous (or T2) generation of transformed plants, and the T2 plants subsequently propagated by classical breeding techniques. A dominant selectable marker (such as nptll) may be associated with the transcription cassette to help ameliorate.

In view of the foregoing, it is obvious that the plants that can be used in the practical embodiment of the present invention include those of the genus Nicotiana.

Specialists familiar with the recombinant DNA methods described above will recognize that to build transgenic tobacco cells and plants, a whole cDNA molecule for QPRT-aase or a complete chromosomal gene for QPRT-ase can be used, in the sense orientation, with the sequences. appropriate operating related regulators. (Experts in the art will also admit that the regulatory sequences suitable for gene expression in targeting orientation include any of the eukaryotic translation initiation sequences, except for the promoter and sequences of polyadenylation / transcription sequences described above). Such transformed tobacco plants are characterized by increased levels of QPRT-azase and, consequently, by higher nicotine contents compared to the non-transformed tobacco control plants.

Therefore, it should be understood that the use of DNA sequences encoding QPRT-aza to lower or increase the levels of the QPRT-aza enzyme, and thus to reduce or increase the nicotine content of tobacco plants, is within the scope of the present invention. inventions.

As used herein, a crop contains a lot of plants of the present invention belonging to the same genus, planted together in an agricultural field. The term agricultural field means a piece of land, or a greenhouse. Thus, the present invention provides a method of producing a plant crop having altered QPRT-azic activity and thus having increased or decreased nicotine levels, compared to a similar crop of non-transformed plants of the same species and variety.

The following are non-limiting examples illustrating the present invention.

Example 1. Isolation and sequencing of the cDNA of TobRD2 gene (Conkling et al., Plant Phys. 93, 1203 (1990)) was sequenced and provided in the present description in the form of SEQ ID NO: 1, and the amino acid sequence was deduced. as SEQ ID NO: 2. The deduced amino acid sequence was assumed to harbor a cytosolic protein. Although no genes for plant QPRT have been reported, comparisons between the amino acid sequence of NtPT1 and the Gene Bank database (fig. 3) revealed a limited sequential similarity with certain bacterial proteins or other proteins; quinolate phosphoribosyl transferase (QPRT-azic) activity has been demonstrated for genes from S. typhimurium, E. coli and N. tabacum. The QPRT-axis encoded by the NtQPTI gene was similar to the peptide fragment deduced and encoded by an Arabidopsis expression sequence tags from Arabidopsis (having access to the F20096 gene bank), which may represent part of the gene for QPRt. -aid from Arabidopsis.

Example 2. In situ hybridizations

To determine the spatial distribution of TobRD2 mRNA transcripts in different root tissues, hybridizations were performed in non-transformed plants. In situ hybridizations of the antisense strand for TobRD2 with the mRNA for TobRD2 in root tissue were performed using techniques such as those described by Meyerowitz, Plant Mol. Biol. Rep. 5,

RO 121968 Β1

242 (1987) and Smith et al., Plant Mol. Biol. Rep. 5, 237 (1987). Roots from 1 tobacco seedlings (Nicotiana tabacum) for 7 days were fixed in phosphate buffered glutaraldehyde, introduced into Paraplast Plus (Monoject Inc., St. Louis, MO) and sectioned at 8 mm 3 thickness to obtain sections. , both transverse and longitudinal. Antisense TobRD2 transcripts, synthesized in vitro in the presence of ATP-35S, were used as 5 probes. The labeled RNA was hydrolyzed by alkaline treatment, with a mass of medium length being obtained, before use, of 100 - 200 bases. 7

Hybridizations were made in 50% formamide for 16 hours at 42 ° C with labeled RNA

- about 5 x 10⁶ units per minute (cpm = "counts-per-minutes") per milliliter of the 9 hybridization solution. After exposure, the blades were developed and visualized by optical microscopy (in illuminated and non-illuminated field). 11

The hybridization signal was located at the level of the cortical layer of the root cells (the results are not shown). Comparison of the images in the illuminated and non-illuminated field 13 of the same sections led to the localization of the transcripts TobRD2 in the parenchymal cells of the root cortex. No hybridization signal was visible in the epidermis or sternum.

Example 3. TobRD2 mRNA levels corresponding in No 17 and / or Nic2 tobacco mutants and their correlation with nicotine levels

The stationary levels of the mRNA corresponding to the TobRD2 19 gene in mutant tobacco plants Nici and Nic2 were examined. It is known that the genes Neither and Nic2 regulate the activity of quinolate phosphoribosyl transferase and the activity of methyl transferase rot and 21 are co-dominant regulators of nicotine production. The present results are illustrated in Figures 5A and 5B and show that the expression of the TobRD2 gene is regulated by Neither and Nic2. 2. 3

RNA was isolated from: Burley 21 wild-type tobacco plant roots (Nic1 / Nic1 Nic2 / Nic2); roots of Nic1-Burley 21 (nic1 / nic1 Nic2 / Nic2); roots of 25 Nic2-Burley 21 (Nic1 / Nic1 nic2 / nic2) and roots of Nic1-Nic2-Burley 21 (neither / nor nic2 / nic2). 27

Four Burley 21 (nic) tobacco lines were grown in the soil for one month, which were transferred to hydroponic chambers in aerated nutritional solution, in the greenhouse, for 29 months. These lines were isogenic, except for the two loci corresponding to the low nicotine level and had the genotypes Nic1 / Nic1 Nic2 / Nic2, Neither / Neither nic2 / nic2, nic1 / nic1 31

Nic2 / Nic2, nic1 / nic1 nic2 / nic2). Roots from approximately 20 plants for each genotype were collected and pooled for RNA isolation. Total RNA (1 µg) from each 33 genotypes was electrophoresed in a 1% agarose gel containing 1.1 M formaldehyde and transferred to a nylon membrane, according to Sambrook et al., (1989) . The memos were hybridized with cDNA fragments for TobRD2, labeled with P32.

The relative intensity of TobRD2 transcripts was measured by densitometry. Figure 5 37 (solid bars) illustrates the relative levels of the transcript (compared to None / No Nic2 / Nic2) for each of the 4 genotypes. The relative nicotine content (compared to Nic1 / Nic1 39 Nic2 / Nic2) of the 4 genotypes is represented by hatched bars.

Fig. 5 graphically compares the relative level, of the steady state, of the mRNA corresponding to the 41 TobRD2 gene, using as a reference amount the level found in the wild type Burley 21 (Nic1 / Nic1 Nic2 / Nic2). TobRD2 mRNA levels corresponding to 43 Nic1 / Nic2 double mutants were approximately 25% of those of wild-type tobacco. Fig. 5B further compares the relative levels of nicotine in the nearly isogenic tobacco lines studied 45 in this example (solid bars indicate the level of TobRD2 transcript; bars bared indicate nicotine levels). There was a close correlation between nicotine levels and levels 47 of the corresponding TobRD2 transcript.

RO 121968 Β1

Example 4. Effect of apical cutting on the levels of mRNA corresponding to TobRD2.

It is well known in the art that removing the floral end of a plant (sausage) accelerates root growth and increases the nicotine content of the leaves of that plant. The sausage of the plant is a standard practice in the cultivation of commercial tobacco, and the optimal time for the sausage of a certain tobacco plant, in a known set of growing conditions, can be quickly determined by a connoisseur in the field.

Tobacco plants (N. tabacum SR1) were obtained from seeds, which were grown in the soil for one month, then transferred to sand pots. The plants were grown in the greenhouse for another 2 months, until flowering began. The flower heads and 2 more knots were removed from 4 plants (sausages). After the indicated time, a portion of the roots of each plant was collected which were collected for the purpose of extracting the RNA. The control plants were not decapitated. Total RNA (1 µg) at each time point was electrophoresed on a 1% agarose gel containing 1.1 M formaldehyde, then transferred to a nylon membrane, as recommended by Sambrookși et al., (1989) . Membranes were hybridized with P-labeled TobRD2 cDNA fragments³². The relative intensity of TBRD2 transcripts was measured by densitometry. Fig. 6 illustrates the relative levels of the transcript (compared to the zero moment) for each moment of time, with sausage (solid bars), or without sausage (hatched bars).

Relative levels of TobRD2 in root tissue after 24 h were determined; the results are shown in FIG. 6 (solid bars indicate TobRD2 transcript levels in sausage plants; dashed bars indicate TobRD2 transcript levels in unfocused controls). Within 6 hours of tobacco plant mating, TobRD2 mRNA levels increased approximately 8-fold in sausage plants; after the same period of time, no growth was observed in the control plants.

Example 5. Complement of the QPRT-deficient bacterial mutant with DNA having SEQ ID NO: 1

The TH265 strain of Escherichia coli is a mutant deficient in quinolate phosphoribosyl transferase (nadC) which, as a result, cannot grow in environments lacking nicotinic acid.

TH265 cells were transformed with an expression vector (pWS161) containing DNA with SEQ ID NO: 1, or transformed only with the expression vector (pKK233). The growth of the transformed bacterium was compared with the growth of the TH265 transformants (pKK233) and with the growth of the untransformed mutant TH265 nadC. The growth was compared on minimal ME medium (without nicotinic acid) with that on minimal ME medium with nicotinic acid addition.

E. coli strain TH265 with QPTase mutation (nadC) was kindly provided by Dr. KT. Huges (Huges et al., J. Bact. 175: 479 (1993). Cells were stored on LB medium and competent cells were prepared according to the method of Sambrook et al. (1989). An expression plasmid was constructed. In pKK2233 (Brosius, 1984) with the cDNA for TobRD2 cloned under the control of the Tac promoter. The resulting plasmid pWS161 was transformed into TH265 cells. The transformed cells were then passed onto agar plates containing minimal medium (Vogel and Bonner, 1956) with or without nicotinic acid (0.0002%) as a supplement. Single TH265 cells and TH265 cells transformed with pKK2233 were passed onto similar plates to be used as controls.

The results are shown in fig. 4. Only DNA-transformed TH265 cells of SEQ ID NO: 1 were grown on nicotinic acid-free medium. These results show that expression of DNA having SEQ ID NO: 1 in TH265 bacterial cells conferred the NadC phenotype on these cells.⁺, confirming that this sequence encodes QPRT-aza. The TobRD2 nomenclature was thus changed to NtQPTI.

RO 121968 Β1

Example 6. Transformation of tobacco plants 1

DNA having SEQ ID NO: 1, in antisense orientation, is operatively linked to a plant promoter (CaMV 35S or the TobRD2 promoter specific to the root cortex) for 3 to produce two different DNA cassettes: CaMV35S promoter / SEQ ID NO: 1 antisense and promoter TobRD2 / SEQ ID NO: 1 antisense. 5

A wild-type tobacco line and a nicotine-reduced tobacco line were selected for transformation, for example, the Burley 21 wild-type 7 (No + / Nic2 +) and the Burley 21 homozygous nic1- / nic2- line. Lots of tobacco cells from each line were transformed using each DNA cassette. The transformation was performed using an Agrobacterium vector, for example a binary vector of Agrobacterium carrying the Ti-border sequences and the nptll gene (which confers resistance to 11 kanamycin and under the control of the nos promoter (npt II)).

The transformed cells are selected and regenerated in transgenic tobacco plants 13 (R_a). Plants are grown to maturity and tested for nicotine content; a subset of transformed tobacco plants has significantly lower nicotine levels, compared to the untreated control plants.

The R plants are then regenerated_a, and transgender segregation is analyzed within the next 17 generations, R ,. The Ri generation is grown to maturity and regenerated; transgender segregation among R offspring₂ indicates which R. plants, are homozygous for transgene 19.

SEQUENCE LIST

I (1) GENERAL INFORMATION (i) APPLICANT: Conkling, Mark A.25

Well, Nandini

Song. Wen27 (ii) TITLE OF THE INVENTION: Adjustment of the expression of quinolate phosphoribosyl transferase (iii) NUMBER OF SEQUENCES: 429 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESS: Kenneth Sibley, BellSeltzer Park & ​​Gibson31 (B) Post Office Drawer 34009 (C) CITY: Charlotte33 (D) STATE: North Carolina (E) CONTRY: USA35 (F) ZIP: 28234 (v) SUB CARE FORM CAN BE READ INFORMATION37 (A) INFORMATION SUPPORT MEDIA: Floppy disk (B) COMPUTER: IBM PC compatible39 (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patent In Release # 1.0, Version # 1.3041 (vi) CURRENT DATA FOR PATENT APPLICATION:

(A) APPLICATION NUMBER: 43 (B) REGISTRATION DATE:

(C) CLASSIFICATION: 45 (viii) INFORMATION RELATING TO THE POWDER / AGENT (A) NAME: Sibley, Kenneth D.47 (B) REGISTRATION NUMBER: 31,665

EN 121968 Β1 (C) REFERENCE NUMBER / ROL: 5051-338P (ix) TELECOMMUNICATION INFORMATION:

(A) PHONE: 919-420-2200 (B) TELEFAX: 919-881-3175 (2) INFORMATION RELATING TO SECTION. ID. NO. 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1399 base pairs (B) TYPE: nucleic acid (C) CHAIN: mono (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) FEATURE:

(A) NAME / CODE: CDS (B) LOCATION: 52..1104 (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID. NUMBER 1

RO 121968 Β1

CAAAAACTAT TTTCCACAM ATTCATîTCA CAACCCCCCC AAMMAAAC C ATG ΤΠ 57 Het Phe

AGA GCT ATT CCT TTC ACT GCT ACA GTG CAT CCT TAT GCA ATT ACA GCT 105 Arg Ala 11 «Pro Phe Thr Ala Thr Val Hts Pro Tyr Ala De Thr Ala

1015

CCA AGG KG GTG GTG AAA ATG TCA GCA ATA GCC ACC AAG AAT ACA AGA153

Pro Arg leu Val Val Lys Met Ser Ala Ile A1a Thr Lys Asn PirArg

2530

6Π3 GAG TCA TTA GAG GTG AAA CCA CCA GCA CAC.CCA ACT TAT GAT TTA201

Val Glu Ser Leu Glu Val Lys Pro Pro Ala His Pro Thr Tyr AspLeu

40 4550

AAG GAA GTT ATG AM CIT GCA CTC TCT GAA GAT GCT GGG MT TTA GGA249

Lys Glu Val Met Lys Leu Ala Leu Ser Glu Asp Ala Gly Asn LeuGly

6065

GAT GTG ACT TGT AAG GCG ACA ATT CCT CTT GAT ATG GAA TCC GAT GCT297

Asp Val Thr Cys Lys Ala Thr Ile Pro Leu Asp Mat Glu Ser AspAla

7580

CAT TTT CTA GCA AAG GAA GAC GGG ATC ATA GCA GGA ATT GCA CTT GCT345

His Phe Leu Ala lys Glu Asp Gly Hair Ile Ala Gly 11 «Ala LeuAla

9095

GAG ATG ATA CTC GCG GAA GTT GAT CCT TCA TTA AAG GTG GAG TGG TAT393

Glu Het Ile Phe Ala Glu Val Asp Pro Ser Leu Lys Val Glu TrpTyr

100 10S110

GTA AAT GAT GGC GAT AAA GTT OT AM GGC TTG AAA TTT GGC AAA GTA441

Val Asn Asp Gly Asp Lys Val His Lys Gly leu Lys Phe Gly Lys Val

115 120 125230

CM GGA AAC GCT TAC MC ATT GTT ATA GCT GAS AGG GTT GTT CTC ΜΓ489

Gin Gly Asn Ala Tyr Asn Ile Val Ile Ala Glu Arg Val Val Leu Asn

135 140145

TTT ATG CM AGA ATG AST. GGA ATA GCT ACA CTA ACT AAG GAA ATG GCA 537 Phe Het Gin Arg Het Ser Gly Ile Ala Tir Leu Thr Lys Glu Het Ala

150 155160

GAT GCT GCA CAC CCT GCT TAC ATC TTC GAS ACT AGG AAA ACT GCT CCT 585 Asp Ala Ala H1s Pro Ala Tyr Ile Leu Glu Thr Arg Lys Thr Ala Pro

165 170175

RO 121968 Β1

GGA ΠΑ CGT TTG GTG GAT AAA TGG GCG GTA TTG ATC GGT GGG GGG AAG

Gly Leu Arg Leu Val Asp Lys lrp ala Val Leu Gly Hair Gly GlyLys

180 185190

MT CAC AGA ATG GGC TTA TTT GAT ATG GTA ATG ATA AM GAC AATCAC

Asn His Arg Met Gly Leu Phe Asp Met Val Met Ile Lys Asp AsnHis

195 200 205210

ATA TCT GCT GCT GGA GGT GTC GGC AAA GCT CTA MA TCT GTG GATCAG

Hair Ser Ala Ala Gly Gly Val Gly Lys Ala Leu Lys Ser Val AspGin

215 220225

T AT TTG GAG CM MT AAA CTT CM ATA GGG GTT GAG GTT GAA ACCAGG

Tyr Leu Glu Gin Asn Lys Leu Gin Ile Gly Val Glu Val Glu ThrArg

230 235240

ACA ATT GAA GAA GTA CGT GAG GTT CTA GAC TAT GCA TCT CM ACAAAG

Thr Ile Glu Glu Val Arg Glu Val Leu Asp Tyr Ala Ser Gin ThrLys

245 258255

ACT TCG TTC ACT AfiG ATA ATC CTG GAC AAT ATC GTT GTT CCA TTATCT

Thr Ser Leu Thr Arg II and Met Leu Asp Asn Met Val Val Pro LeuSer

260 265270

AAC GGA GAT ATT GAT GTA TCC ATG CTT AAG GAG GCT GTA GAA TTGATC

Asn Gly Asp Ile Asp Val Ser Met Leu Lys Glu Ala Val Glu LeuIle

275 280 285290

MT GGG AGG TTT GAT ACG GAG GCT TCA GGA AAT GTT ACC CTT GAAACA

Asn Gly Arg Phe Asp Thr Glu Ala Ser Gly Asn Val Thr Leu GluThr

295 300305

GTA CAC MG ATT GGA CAA ACT GGT CTT ACC TAC ATT TCT AGT GGTGCC

Val His Lys Ile Gly Gin Thr Gly Val Thr Tyr Ile Ser Ser GlyAla

310 315320

CTC ACG CAT TCC GTG AAA GCA CTT GAC ATT TCC CTG MG ATC GATACA

Leu Thr His Ser Val Lys Ala Leu Asp Ile Ser Leu Lys They AspThr

325 330335

GAG CTC GCC CTT GAA GTT GGA AGG CGT ACA AM CGA GCA TGAGCGCCAT Glu Leu Ala Leu Glu Val Glv Arg Arg Thr Thr Lys Arg Ala

340 345350

TAOTCTGCT ATAGGGTTGG AGTAAMGCA GCTCMTASC TGAMfiGTCC MATAAGAAT CATîTTACTA GTTCTCAAAC AMAGATCCT TCACTGTGTA ATCAAACMA AAfîATGTAAA TTCCTGGMT ATCTCAGATG GCTCTTTTCC MCCTTATTG ClTCAfîTTGG ΤΑΑΤΓΤΟΑΠ ATAGCTHGT TTTCATCTTT CATBGAATÎT GTTACAATGA AMTACITGA TTTATAAGTT TGOTCTATCT MAATTCTGT CTTACTTCM ATATTTTGAG ATGTT

633

681

729

777

825

873

921

969

1017

1065

1114

1174

1234

1294

1354

1399

RO 121968 Β1 (2) INFORMATION CONCERNING SECTION. ID. NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID. NO: 2

Het Phe Arg Ala Hair Pro Phe Thr Ala Thr / al His Pro Tyr Ala Ile 15 1015

Thr Ala Pro Arg Leu Val Val Lys Met Ser Ala Ile Ala Thr Lys Asn 20 2530

Thr Arg Val Glu Ser Leu Glu Val Lys Pro Pro Ala HIs Pro Thr Tyr 35 4045

Asp Leu Lys Glu Val Met Lys Leu Ala Leu Ser Glu Asq Ala Gly Asn 50 5560

Leu Gly Asp Val Thr Cys Lys Ala Thr ile Pjo Leu Asp Met Glu Ser 65 70 7580

Asp AU His Phe Leu Ala Lys Glu Asp Gly Ile Hair Ala Gly Hair Ah 85 9095

Leu Ah Glu Met He Phe Ala Glu Val Asp Pro Ser Leu Lys Val Glu 100 105110

Trp Tyr Val Asn Asp Gly Asp Lys Val His lys Gly Leu Lys Phe Gly 115 120125

Lys Val Gin Gly Asn Ala Tyr Asn Ile Val Ile Al a Glu Arg Val Val 130 135140

Leu Asn Phe Met Gin Arg Met Ser Gly Ile Ah Thr Leu Thr lysGlu

145 150 155160

Hey Ala Asp Ala Ala Hls Pro Ala Tyr Ile Leu Glu Thr Arg LysThr

165 170175

Ah Pro Gly Leu Arg Leu Val Asp Lys Trp Ah Val Leu Gly Gly Hair 160 185190

Gly Lys Asn His Arg Met'Gly leu Phe Asp Met Val Met Ile Lys Asp 195 200205

Asn His Ile Ser Ala Ala Gly Gly Val Gly Lys Ala Leu Lys Ser Val 210 215220

Asp Gîn Tyr Leu Glu Gin Asn Lys Leu Gin He Gly Val Glu Val Glu 225 230 235240

RO 121968 Β1

<td>Ttir</td><td rowspan="2">Arg</td><td>Thr</td><td>Ile</td><td>Glu</td><td>Glu</td><td>Wave</td><td rowspan="2">Arg</td><td>Glu</td><td>Wave</td><td>Lion</td><td rowspan="2">Asp</td><td>Tyr</td><td>That</td><td>Serum</td><td>Gin</td>

<td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td> 255</td><td></td>

<td>B.C</td><td rowspan="2">Lys</td><td>Thr</td><td>Serum</td><td>Lion</td><td>Thr</td><td rowspan="2">Arg</td><td>us</td><td>Het</td><td>Lion</td><td rowspan="2">Asp</td><td>Asn</td><td>Het</td><td>Wave</td><td>Wave</td><td>Pro</td>

<td></td><td></td><td> 260</td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Lion</td><td>Serum</td><td>Asn</td><td rowspan="2">Gly</td><td rowspan="2">Asp</td><td>Ile</td><td rowspan="2">Asp</td><td>Wave</td><td>Serum</td><td>Het</td><td>lion</td><td rowspan="2">Lys</td><td>Glu</td><td>That</td><td>Wave</td><td>Glu</td>

<td></td><td></td><td> 275</td><td></td><td> 280</td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Lion</td><td>ll e</td><td>Asn</td><td>Gly</td><td rowspan="2">Arg</td><td>Phe</td><td>Asp</td><td>Thr</td><td>Glu</td><td>That</td><td>Serum·</td><td>Gly</td><td>Asn</td><td>Wave</td><td>Thr</td><td>Lion</td>

<td></td><td> 290</td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Glu</td><td>ΊΠ1Γ</td><td>See you 1</td><td>l-lis</td><td rowspan="2">Lys</td><td>Ile</td><td rowspan="2">Gly</td><td>Gin</td><td>Thr</td><td rowspan="2">Gly</td><td>wave</td><td>Thr</td><td>Tyr</td><td>Ile</td><td>Serum</td><td>Serum</td>

<td> 305</td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td rowspan="2">Gly</td><td>That</td><td>Lion</td><td>Ί hr</td><td>His</td><td>Serum</td><td>Wave</td><td rowspan="2">Lys</td><td>That</td><td>Lion</td><td rowspan="2">Asp</td><td>Ile</td><td>Serum</td><td>Lion</td><td>Lys</td><td>Ile</td>

<td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Asp</td><td>Thr</td><td>Glu</td><td>Lion 340</td><td>That</td><td>Lion</td><td>GTU</td><td>Wave</td><td>Gly 345</td><td>Arg</td><td>Arg</td><td>Thr</td><td>Lys</td><td>Arg 350</td><td>That</td><td></td>

(2) INFORMATION RELATING TO SEQ. ID. NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1053 base pairs (B) TYPE: nucleic acid (C) CHAIN: mono (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cADN (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID. NO: 3

RO 121968 Β1

ATGTTTAGAG CTATTCCTTT TIGGT6B1GA AAATGTCAGC AAACCACCAG CACACCCAAC GATGCTGGGA ATITAGGAfiA GATGCTCATT TTCTAGCAAA ATATTCGCGG AAGT1GATCC GnCATAAAG GCTTGAAAn GAGAGGGTTG TTCTCAATTT ATGGCAGATG CTGCACACGC CGTlTGGTGG ATAAATGGGC TnGATATGG TAATGATAAA CTAAAATCTG TGGATCAGTA ACCAGGACM HGAAGAAGT nGACTAGGA TAATGCTGGA TCCATGCTTA AGMCTST MTGTTACCC HGAâACAGT GGTGCCCTGA CGCATTCCCT GCCCTTGAAG HGGAASGCG

CACTGCTACA GTGCATCCTT AATAGCCACC AAGAATACAA TTATGATTTA AAGGAAGTTA TGTGACTTBT AAGGCGACAA GGAAGACGGG AICATAGCAG nCAUAAAG GTGGAGTGGT TGGCAAAGTA CAAGGAAACG TATGCAAAGA ATGAGTGGM TGCT1ACATC HGGAGACTA GGTAKGATC ggtgggggga AGACAATCAC ATATCTGCTG TÎTGGAGCAA AAÎAAACTfC ACGTGAGGTŢ CTAGACTATR CAATATGGTŢ GHTCCAITAT AGAATTCATC AAT6GGAGGT ACACAAGATT GGACAMCTG GAAAGCACTT GACATTTCCC TACAAAACGA GCA

ATGC / WTAC AGCTCCAAGG GAGTGGAGTC AflAGAOGlG TGAAACTTGC ACrCTCTGAA TTCCTCTTGA TATGGAATCC GAAHGCACT TGCTGAGATG ATGTAAATGA TGGCGATAAA CTTACAACAT TGTTATAGTA TAAGCTACAAC. GGAAAACTGC TCCTSSAHA AGAATCACAG AATGGGGCTÎA CTGGAGG1G1 CGGCAAAGCT AAATAGGGGT TGAGGTFGAA CA7CTCAAAC AAAGACTTCG CTAACGGAGA TAITGATCTA UGATACGGA GGCTTCAGGA GTGTTACCTA

<td colspan="2"> 60</td>

<td>L20</td><td> 3</td>

<td> 180</td><td> 5</td>

<td> 240</td><td></td>

<td> 300</td><td> 7</td>

<td> 360</td><td> 9</td>

<td> 420</td><td></td>

<td> 480</td><td> 11</td>

<td> 540</td><td> 13</td>

<td> 600</td><td></td>

<td> 650</td><td> 15</td>

<td> 720</td><td> 17</td>

<td> 780</td><td></td>

<td> 340</td><td> 19</td>

<td> 900</td><td> 21</td>

<td> 960</td><td rowspan="2"> 23</td>

<td> 1020</td>

<td> 1053</td><td> 25</td>