MX2007010520A - Reducing levels of nicotinic alkaloids in plants - Google Patents

Abstract

Two genes, A622 and NBBl, can be influenced to achieve a decrease of nicotinic alkaloid levels in plants. In particular, suppression of one or both of A622 and NBBl may be used to decrease nicotine in tobacco plants.

Description

REDUCTION OF LEVELS OF NICOTINIC ALKALOIDS IN PLANTS FIELD OF THE INVENTION The present invention relates to the field of molecular biology and the sub-regulation of alkaloid synthesis. More specifically, the invention relates to the methodology and constructs for the reduction of nicotinic alkaloids in a plant, particularly but not exclusively in a tobacco plant.

BACKGROUND OF THE INVENTION Currently, there are several methods for the reduction of nicotinic alkaloids, such as nicotine, in plants. A low nicotine strain of tobacco has been used, for example, as a breeder for low nicotine cultures. Legg et al., Crop Sci 10: 212 (1970). Genetic engineering methods can also be used to reduce nicotine levels. For example, U.S. Patent Nos. 5,369,023 and 5,260,205 describe the decrease in nicotine levels via the antisense direction of an endogenous putrescine methyltransferase (PMT) sequence. Voelckel et al., Chemoecology 11: 121-126 (2001). The tobacco quinolate phosphoribosyl transferase (QPT) gene has been cloned, Sinclair et al., Plant Mol. Biol. 44: 603-617 (2000), and its antisense suppression Ref. : 185760 provided significant nicotine reductions in transgenic tobacco plants. Xie et al., Recent Advances in Tobacco Science 30: 17-37 (2004). See also the patents of E.U.A. Nos. 6,586,661 and 6,423,520. Several enzymes of nicotine biosthesis are known.

For example, see Hashimoto et al., Plant Mol. Biol. 37: 25-37 (1998); Reichers & Timko, Plant Mol. Biol. 41: 387-401 (1999); Imanishi et al., Plant Mol. Biol. 38: 1101-1111 (1998). Still, there is a continuing need for additional genetic engineering methods for further reduction of nicotinic alkaloids. When only PMT is sub-regulated in tobacco, for example, nicotine is reduced but anatabine is increased by about 2 to 6 times. Chintapakorn & Hamill, Plant Mol. Biol 53: 87-105 (2003); Steppuhn, et al., PLoS Biol 2 (8): e217: 1074-1080 (2004). When only the QPT is sub-regulated, an abundant amount of alkaloids remains. See U.S. Plant Variety Certificate No. 200100039. The total reduction of the content of alkaloids in tobacco would increase the value of tobacco as a source of biomass. When grown under conditions that maximize biomass, such as high density and multiple cuts, tobacco can produce more than 8 tons in dry weight per acre, which is verifiable with other crops used for biomass. However, the large-scale growth and processing of conventional tobacco biomass has several disadvantages. For example, significant time and energy are spent in the extraction, isolation, and elimination of tobacco alkaloids because the conventional tobacco biomass that depends on the variety contains about 1 to 5 percent alkaloids. On a per-acre basis, conventional tobacco biomass contains approximately as much as 800 pounds (400 kg) of alkaloids. Also, people who handle tobacco may suffer from overexposure to nicotine, commonly referred to as "green tobacco sickness." Reduced alkaloid tobacco is more susceptible for non-traditional purposes, such as biomass and derived products. For example, it is advantageous to use reduced alkaloid tobacco for the production of ethanol and protein co-products. The application published in US No. 2002/0197688. Additionally, alkaloid-free tobacco or fractions thereof may be used as a forage crop, animal feed, or a human nutrient source. Id. Beyond these benefits associated with nicotine reduction, more successful methods are needed to help smokers in the process of quitting. Nicotine replacement therapy (NRT) is not very effective as a treatment to stop smoking because its success rate is less than 20 percent after 6 to 12 months from the end of the nicotine replacement period. Bohadana et al., Arch Intern. Med. 160: 3128-3134 (2000); Croghan et al., Nicotine Tobacco Res. 5: 181-187 (2003); Stapleton et al., Addiction 90: 31-42 (1995). Nicotine-reduced or nicotine-free tobacco cigarettes have assisted smokers in the process of successfully quitting, by leaving out the nicotine smoker still allowing the smoker to perform the smoking ritual. Additionally, de-nicotinized cigarettes relieve cravings and other symptoms of smoking abstinence. See Rose, Psychopharmacology 184: 274-285 (2006) and Rose et al., Nicotine Tobacco Res. 8: 89-101 (2006). Accordingly, there is a continuing need to identify additional genes whose expression may be affected to decrease the nicotinic alkaloid content.

BRIEF DESCRIPTION OF THE INVENTION Two genes, A622 and NBBl, can be influenced to achieve a decrease in nicotinic alkaloid levels in plants. In particular, the deletion of one or both of A622 and NBB1 can be used to decrease nicotine in tobacco plants. Accordingly, in one aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from (a) nucleotide sequence set forth in SEQ ID NO: 3; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, and (c) a nucleotide sequence that differs from the nucleotide sequences of (a) or (b) due to to the degeneracy of the genetic code and encodes a polypeptide with NBBl expression. In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from (a) a nucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; and (c) a nucleotide sequence that differs from the nucleotide sequences of (a) or (b) due to the degeneracy of the genetic code and encodes a polypeptide with A622 expression, wherein the nucleotide sequence is operably linked to a promoter. heterologous In another aspect, the invention provides a method for the reduction of an alkaloid in a plant, comprising decreasing the expression of NBBl and A622. In another aspect, the invention provides a transgenic plant having alkaloid content and reduced A622 expression, in addition a tobacco plant having NBB1 expression and reduced alkaloid content. The invention also provides a genetically engineered plant having reduced nicotine and anatabine content. In another aspect, the invention provides a reduced nicotine tobacco product made from a tobacco plant that has reduced A622 or NBBl expression.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1 describes the nicotine biosynthesis pathway. The abbreviations are: AO = aspartate oxidase, QS = quinolinate synthase, QPT = phosphoribosyl transferase quinolinate, ODC = ornithine decarboxylase, PMT = N-methyltransferase of putrescine, and DAO = diamine oxidase. FIGURE 2A schematically illustrates pHANNIBAL. FIGURE 2B schematically illustrates pHANNIBAL-X in which the multiple ligation sites have been modified. FIGURE 3 depicts the scheme for the preparation of a binary plant RNAi vector using the modified pHANNIBAL-X as an intermediate plasmid. FIGURE 4 represents the T-DNA region of pR Ai-A622. FIGURE 5 depicts the accumulation of nicotine alkaloid in BY-2 cells, silenced BY-2 cells of A662, and silenced BY-2 cells of NBBl. FIGURE 6 depicts the expression of A622, NBB1, and genes for unknown enzymes in the nicotine biosynthesis pathway in wild type BY-2 cells, silenced BY-2 cells of A662, and silenced BY-2 cells of NBB1. FIGURE 7 depicts the T-DNA region of the inducible A622 expression vector pXVE-A622RNAi. FIGURE 8A depicts the specific suppression of A622 in hairy root lines transformed with an inducible A622 deletion construct after suppression of induction with estradiol. FIGURE 8B illustrates the reduced nicotine content in hairy root lines transformed with an inducible A622 deletion construct after induction of suppression with estradiol. FIGURE 9 depicts RNA staining analysis of NBBl expression and expression of PMT in root and leaf tissue of wild-type tobacco and mutants of niel, nic2, and niclnic2. FIGURE 10 depicts an alignment of NBB1 with berberine bridge enzyme from Eschscholzia californica (EcBBE). FIGURE 11 depicts a phylogenetic tree constructed using protein sequences similar to plant BBE and NBBl. FIGURE 12 represents the T-DNA region of the NBBl suppression vector pHANNIBAL-NBB1 3 '. FIGURE 13 represents the reduction of nicotinic alkaloid synthesis in hairy root of snuff suppressed NBB1. FIGURE 14 represents the expression of NBBl, A622, and known enzymes involved in nicotine biosynthesis in silenced hairline lines of NBBl and control. FIGURE 15 represents the T-DNA region of the NBBl deletion vector of complete pANDA-NBB1. FIGURE 16 depicts nicotine levels in the leaves of Nicotiana tabacum plants of lines transformed with the NBBl deletion vector of complete pANDA-NBB1.

DETAILED DESCRIPTION OF THE INVENTION The present invention identified two genes, A622 and NBB1, which can be influenced to achieve a decrease in nicotine alkaloid levels in plants including but not limited to tobacco. While A622 was previously identified by Hibi et al. Plant Cell 6: 723-735 (1994), the present inventors discovered a role for A622, hitherto unknown, in the context of decreased nicotine biosynthesis. The field was completely unaware of NBB1, before the discovery of the inventor, and also elucidated a role for NBBl in an approach, in accordance with the present invention, for reducing the content of nicotinic alkaloid in plants. Accordingly, the present invention encompasses both the methodology and the constructs for reducing the content of nicotinic alkaloid in plant, by suppressing the expression of A622 or NBBl. That is, nicotinic alkaloid levels can be reduced by suppressing one or both of A622 and NBBl. According to this aspect of the invention, a plant or any part of it is transformed with a nucleotide sequence, the expression of which suppresses at least one of A622 and NBB1 and reduces the content of nicotinic alkaloid. In another aspect of the invention, nicotine can be further suppressed in a plant by the expression of concurrent deletion of any enzyme in the nicotine biosynthetic pathway, such as QPT or PMT, and at least one of A622 and NBBl. In addition to decrease nicotine, for example, the present invention provides means for concurrent reduction of anatabine. Thus, anatabine levels can be decreased by suppression of a nicotine biosynthesis gene, such as QPT, and at least one of A622 and NBBl. Through the affectation of the expression of A622 and / or NBBl, to the terminations of reduction of the content of nicotinic alkaloid in a plant, numerous plants of reduced alkaloids and by-products can be obtained, maintaining the present invention. For example, a tobacco plant having expression of A622 and suppressed NBBl can be used for the production of reduced nicotine cigarettes, which may find use as a smoking cessation product. In the same way, reduced nicotine tobacco can be used as a fodder crop, animal feed, or a source for human nutrition. Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since changes and modifications within the spirit and scope of the invention may become palpable for those skilled in the art from this detailed description.

Definitions The technical terms used in this specification are commonly used in biochemistry, molecular biology and agriculture; hence, they are understandable by those experts in the field to which this invention pertains. Those technical terms can be found, for example, in: MOLECULAR CLONING: A LABORATORY MANUAL, 3rd ed. , vol. 1-3, ed. Sambrook and Russel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, ed. Ausubely collaborators, Greene Publishing Associates and Wiley-Interscience, New York, 1988 (with periodic updates); SHORT PROTOCOLS IN MOLECULAR BIOLOGY: A COMPENDIUM OF METHODS FROM CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 5th ed. , vol. 1-2, ed. Ausubel et al., John Wiley & Sons, Inc., 2002; GENOME ANALYSIS: A LABORATORY MANUAL, vol. 1- 2, ed. Green et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1997. The methodology that involves plant biology techniques is described herein and described in detail in methodology treatises such as METHODS IN PLANT MOLECULAR BIOLOGY: A LABORATORY COURSE MANUAL, ed. Maliga et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1995. Several techniques using PCR are described, for example, in Innis et al., PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press, San Diego, 1990 and in Dieffenbach and Dveksler, PCR PRIMER: A LABORATORY MANUAL, 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2003. The PCR primer pairs can be derived from known sequences by known techniques such as those using computer programs projected for that purpose, for example, Primer, Version 0. 5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA. Methods for the chemical synthesis of nucleic acids are described, for example, in Beaucage & amp;; Caruthers, Tetra. Letts. 22: 1859-1862 (1981), and Matteucci & Caruthers, J. Am. Chem. Soc. 103: 3185 (1981). The digestions, phosphorylations, ligatures and transformations of restriction enzymes were made as described in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989), Cold Spring Harbor Laboratory Press. All reagents and materials used for growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), Invitrogen (Gaithersburg, Md.), Or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified. The expression of A622 is controlled by the location of the NIC1 and NIC2 gene in the tobacco plants. Hibi et al., The Plant Cell, 6: 723-735 (1994). It has been reported that the A622 exhibits the same expression pattern as PMT. Shoji, T. et al., Plant Cell Physiol, 41: 9: 1072-1076 (2000a); Shoji,. , and collaborators, Plant Mol Biol, 50: 427-440 (2002). Both A622 and PMT are specifically expressed in roots, particularly in the cortex and endoderms of the apical parts of roots and root hairs. Shoji et al. (2002). Furthermore, A622 and PMT have a common pattern of expression in response to NIC regulation and methyl jasmonate stimulation. The A622 is induced in the roots of Nicotiana tabacum in response to the injury of the aerial tissues. Cañe et al., Functional Plant Biology, 32, 305-320 (2005). In N. glauca, A622 is induced in injured leaves under conditions that result in induction of QPT. Sinclair et al., Func. Plant Biol., 31: 721-9 (2004). The nucleic acid sequence of A662 (SEQ ID NO: 1) has been determined. Hibi et al. (1994), supra. The protein encoded by this nucleic acid sequence (SEQ ID NO: 2) resembles the isoflavone reductases (IFR) and contains an NADPH binding moiety. A622 shows homology with TP7, a tobacco phenylcoumaran benzyl ether reductase (PCBER) involved in lignin biosynthesis. Shoji et al. (2002), supra. No PCBER activity was observed, however, when A622 was expressed in E. coli it was tested with two different substrates. Based on the co-regulation of A622 and PMT and the similarity of A622 to IFR, A622 was proposed to function as a reductase in the final steps of the alkaloid synthesis. Hibi et al. (1994); Shoji, et al. (2000a). However, IFR activity was not observed, when the protein was expressed in bacteria (id.). The function of A622 was previously unknown, and it was not understood until then that A622 plays a role in the synthesis of nicotine. The expression of A622 refers to biosynthesis of a gene product encoded by SEQ ID N0: 1. The deletion of? 622 refers to the reduction of the A622 expression. The expression A622 has an ability to regulate the content of nicotonic alkaloid in a plant or plant cell. An alkaloid is a basic compound that contains nitrogen found in plants and produced by secondary metabolism. A nicotine alkaloid is nicotine or an alkaloid that is structurally related to nicotine and that is synthesized from a compound produced in the nicotine biosynthesis pathway. In the case of tobacco, the nicotine alkaloid content and the total alkaloid content are used synonymously. Illustrative Nicotiana alkaloids include but are not limited to nicotine, nornicotine, anatabine, anabasine, anatalin, N-methylanatabine, N-methylanabasine, myosmin, anabaseine, N '-formilnornicotine, nicotirine, and cotinine. Other minor alkaloids are reported on the tobacco leaf, for example, in Hecht, S. S. et al., Accounts of Chemical Research 12: 92-98 (1979); Tso, T.C., Production, Physiology and Biochemistry of Tobacco Plant. Ideáis Inc., Beltsville, MD (1990). The chemical structures of several alkaloids are present, for example, in Felpin et al., J. Org. Chem. 66: 6305-6312 (2001). Nicotine is the primary alkaloid in N. tabacum along with 50-60 percent of other Nicotiana species. Based on the accumulation of alkaloid in the leaves, nicotine, anatabine, and anabasine are the first alkaloids in N. tabacum. Anatabine is usually not the primary alkaloid in any species, but it accumulates in relatively high amounts in 3 species; Anabasin is the primary alkaloid in four species. Nicotine is the primary alkaloid in 30 to 40 percent of Nicotine species. Depending on the variety, about 85 to about 95 percent of the total alkaloids in N. tabacum is nicotine. Bush, L.P., Tobacco Production, Chemistry and Technology, Coresta 285-291 (1999); Hoffmann, et al., Journal of Toxicology and Environmental Health, 41: 1-52, (1994). In the present invention, the content of the nicotonic alkaloid can be reduced in a genetically engineered plant by regulation of at least one of A622 and NBBl. Additionally, a content of nicotinic alkaloid can be decreased by regulation of a nicotine biosynthesis enzyme, such as QPT or PMT, and at least one of A622 and NBBl. Anatabine is a nicotinic alkaloid. Previous studies have shown that the suppression of PMT reduces the nicotine content but increases the levels of putrescine and anatabine. Chintapakorn & Hamill, Plant Mol. Biol. 53: 87-105 (2003); Sato et al., Proc. NatiAcad. Sci. USA 98, 367-372. (2001); Steppuhn, A., and collaborators, PLoS Biol 2 (8): e217: 1074-1080 (2004). For the purposes of the present invention, the content of anatabine can be decreased in a genetically engineered plant by regulation of at least one of A622 and NBBl. The levels of anatabine can be further decreased by the regulation of a nicotine biosynthesis enzyme, such as QPT, and at least one of A622 and NBBl. A Tobacco Cell BY-2 is a line of cells established in the 60's by Japan Tobacco Co., Ltd. from a variety of Bright Yellow-2 tobacco. Since this cell line grows very fast in tissue culture, it is easy to grow on a large scale and is susceptible to genetic manipulation. BY-2 tobacco cells are widely used as a model plant cell line in basic research. When grown in a standard medium, BY-2 tobacco cells do not produce nicotinic alkaloids. The addition of jasmonate within the culture medium induces the formation of nicotinic alkaloids. Complementary DNA (cDNA) is a single-stranded DNA molecule that is formed from a mRNA template by enzyme reverse transcriptase. Those skilled in the art also use "cDNA" to denote a concatenated DNA molecule that includes such a single-stranded DNA molecule and its complementary DNA strand.

Commonly, a complementary bait for portions of mRNA is used for the initiation of a reverse transcription process that produces a cDNA. Expression refers to the biosynthesis of a gene product. In the case of a structural gene, for example, the expression involves the transcription of the structural gene within mRNA and the translation of the mRNA within one or more polypeptides. "Gene" refers to a polynucleotide sequence comprising the control and coding sequences necessary for the production of a polypeptide or precursor. The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence. A gene may constitute an uninterrupted coding sequence or may include one or more introns, linked by the appropriate spliced linkages. Moreover, a gene may contain one or more modifications either in the coding or non-translation regions that may affect the biological activity or chemical structure of the expression product, the degree of expression, or the way of controlling the expression. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides. In this regard, the modified genes can be referred to as "variants" of the "native" gene. Genetically Designed (GE) covers any methodology for the introduction of a specific nucleic acid or mutation within a host organism. For example, a tobacco plant is genetically engineered when transformed with a polynucleotide sequence that suppresses the expression of a gene, such as A622 or NBB1, and thereby reduces nicotine levels. In contrast, a tobacco plant that is not transformed with a polynucleotide sequence that suppresses the expression of a target gene is a control plant and is referred to as an "untransformed" plant. In the present context, the "genetically engineered" category includes "transgenic" plants and plant cells (see definition, infra), in addition to plants and plant cells produced through directed mutagenesis carried out, for example, through the use of AR / chimeric DNA oligonucleotides, as described by Beetham et al., Proc. Nati Acad. Sci. USA 96: 8774-8778 (1999) and Zhu et al., Loe. cit. at 8768-8773, or so-called "recombinagenic oligonucleobases", as described in PCT application WO 03/013226. In the same way, a genetically engineered plant or plant cells can be produced by introducing a modified virus, which, in turn, causes a genetic modification in the host, with results similar to those produced in a transgenic plant, as described herein. See, for example, U.S. Patent No. 4,407,956. Additionally, a genetically engineered plant or cells can be the product of any native approach (that is, involving non-external nucleotide sequences), implemented by introducing nucleic acid sequences derived only from the host plant species or from sexually compatible plant species. See, for example, published application of US No. 2004/0107455. A Genomic Library is a collection of clones that contains at least one copy of essentially every DNA sequence in the genome. The NBB1 sequence was identified by a cDNA microarray prepared from the cDNA library derived from Nicotiana sylvestris, according to the protocol of Katoh et al., Proc. Japan Acad. , 79 (Ser. B): 151-154 (2003). NBBl is also controlled by the regulatory location of nicotine biosynthesis, NIC1 and NIC2. NBBl and PMT have the same expression pattern in tobacco plants. That NBB1 that is involved in nicotine biosynthesis is indicated by the fact that NBB1, such as PMT and A622, are under the control of the NIC genes and exhibit a similar pattern of expression. The expression of NBB1 refers to the biosynthesis of a gene product encoded by SEQ ID NO: 3. The suppression of NBB1 refers to the reduction of NBB1 expression. The suppression of NBBl has an ability to regulate nicotinic alkaloid content. The location of NIC1 and NIC2 are two independent genetic locations in N. tabacum, formally designated as A and B. Niel and nic2 mutations reduce the expression levels of nicotine biosynthesis enzymes and nicotine content, generally the content of wild type nicotine >; homozygous nic2 > homozygous nel > plants homozygous and nic2 homozygous. Legg & Collins, Can. J. Cyto. 13: 287 (1971); Hibi et al., Plant Cell 6: 723-735 (1994); Reed & Jelesko, Plant Science 167: 1123 (2004). Nicotine is the main alkaloid in N. tabacum and some other species in the Nicotiana gene. Other plants have the ability to produce nicotine, including, for example, the genera Duboisia, Anthocericis and Salpiglessis in the Solanaceae, and the genus Eclipta and Zinnia in the Composites. Plant is a term that encompasses whole plants, plant organs (eg, leaves, stems, roots, etc.), and plant cells and progeny thereof. The plant material includes, without limitation, seed suspension cultures, embryos, meristematic regions, callus tissues, leaves, roots and branches, gametophytes, sporophytes, pollen, and microspores. The class of plants which can be used in the present invention is generally as broad as the class of larger plants susceptible to transformation techniques, including both monocotyledonous and dicotyledonous. A preferred plant is a plant that has nicotine production capacity of the genus Nicotiana, Ouboisia, Anthocericis and Salpiglessis in the Solanaceae or the genus Eclipta and Zinnia in the Composites. A particularly preferred plant is Nicotiana tabacum. Protein refers to a polymer of amino acid residues. A reduced nicotine plant comprises a genetically engineered plant containing less than half, preferably less than 25%, and more preferably less than 20% or less than 10% of the nicotine content of a non-transgenic control plant of the same type. A reduced nicotine plant also includes a genetically engineered plant that contains less total alkaloids compared to a control plant. A "structural gene" refers to a DNA sequence that is transcribed into messenger RNA (mAR) which is then translated into an amino acid sequence characteristic of a specific polypeptide. "Messenger RNA (mRNA)" denotes an RNA molecule that contains the information encoded for the amino acid sequence of a protein. Sequence identity or "identity" in the context of two nucleic acids or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence in a specific region. When the percentage of sequence identity is used in reference to proteins, those residue positions are recognized which are not identical and often differ by conservative amino acid substitutions, where the amino acid residues are substituted for other amino acid residues with similar chemical properties, such as charge and hydrophobicity, and therefore does not change the functional properties of the molecule. Where the sequences differ in conservative substitutions, the percentage of sequence identity can be adjusted upward to correct the conservative nature of the substitution. The sequences which differ from the conservative substitutions are those that have "sequence similarity" or "similarity". The means for making this adjustment are well known to those of skill in the art. Commonly this involves recording a conservative substitution as partial rather than a complete imbalance, thus increasing the percentage of sequence identity. Thus, for example, when an identical amino acid is given a record of 1 and a non-conservative substitution is given a record of zero, a conservative substitution is given a record between zero and 1. The record of conservative substitutions is calculated,. for example, according to the Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17 (1988), as implemented in the PC / GENE program (Intelligenetics, Mountain View, California, USES). The use in this description of a sequence identity percentage denotes a value determined by comparison of two sequences aligned optimally in a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (that is, gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which the identical nucleic acid base or the amino acid residue occurs in both sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence identity. The sequence identity has a recognized significance in the art and can be calculated using published techniques. See COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, ed.

(Oxford University Press, 1988), BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, ed. (Academic Press, 1993), COMPUTER ANALYSIS OF SEQUENCE DATA, PART I5 Griffin & Griffin, eds., (Humana Press, 1994), SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, Von Heinje ed. , Academic Press (1987), SEQUENCE ANALYSIS PRIMER, Gribskov & Devereux, eds. (Macmillan Stockton Press, 1991), and Carillo & Lipton, SIAMJ. Applied Math. 48: 1073 (1988). Methods commonly employed to determine the identity or similarity between two sequences include but are not limited to those described in GUIDE TO HUGE COMPUTERS, Bishop, ed. , (Academic Press, 1994) and Carillo & Lipton, supra. The methods to determine the identity and similarity are codified in computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul and collaborators, J. MoL Biol. 215: 403 (1990)), and FASTDB (Brutlag et al., Comp.App.Biosci., 6: 237 (1990)). Tobacco refers to any plant in the genus of Nicotiana that produces nicotinic alkaloids. Tobacco also refers to products comprising material produced by a Nicotiana plant, and therefore includes, for example, cigarettes, chewable tobacco, snuff and cigarettes made of GE reduced nicotine tobacco for use in smoking cessation. Examples of Nicotiana species include but are not limited to N. alata, N. glauca, N. longiflora, N. persica, N. rustica, N. sylvestris, and N. tabacxm. Tobacco-specific nitrosamines (TSNAs) are a class of carcinogens that are formed predominantly in tobacco during healing, processing and smoking. Hoffman, D., et al., J. Nati Cancer Inst. 58, 1841-4 (1977); Wiernik A et al., Recent Adv. Tob. Sci, (1995), 21: 39-80. TSNAs, such as 4- (N-nitrosomethylamino) -1- (3-pyridyl) -1-butanone (NNK), 1 -nitrosonornicotine (NNN), N'-nitrosoanatabin (NAT), and '-nitrosoanabasin (NAB), they are formed by JV-nicotine nitrosation and other minor Nicotiana alkaloids, such as nornicotine, anatabine, and anabasine. Nicotine reduction alkaloids reduce the level of TSNA in tobacco and tobacco products. The hairy roots of tobacco refer to tobacco roots that have T-DNA from a Ri plasmid of Agrobacterium rhizogenes integrated in the genome and grows in culture without supplementation of auxin and other pitohormones. The hairy roots of tobacco produce nicotinic alkaloids as the roots of a tobacco plant. Transgenic plant refers to a plant that comprises a nucleic acid sequence that is also present per se in another organism or species or that is optimized relative to the host codon of use, from another organism or species. A transgenic plant can be produced by any genetic transformation methodology. Appropriate transformation methods include, for example, Agrobacterium-mediated transformation, particle bombardment, electroporation, polyethylene glycol fusion, transposon labeling, and site-directed mutagenesis. The identification and selection of a transgenic plant are well-known techniques, the details of the needs can not be repeated in the present. A variant is a nucleotide or amino acid sequence that deviates from the standard, or given nucleotide or amino acid sequence of a particular gene or protein. The terms "isoform", "isotype", and "analogue" also refer to "variant" forms of a nucleotide or an amino acid sequence. An amino acid sequence that is altered by the addition, removal or substitution of one or more amino acids, or a change in the nucleotide sequence, can be considered a "variant" sequence. The variant may have "conservative" changes, where a substituted amino acid has similar structural or chemical properties, for example, replacement of leucine with isoleucine. A variant may have "non-conservative" changes, for example, replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residue can be substituted, inserted, or deleted can be found using computer programs well known in the art such as the Vector NTI Suite software (inforMax, MD). The "variant" may also refer to a "mixed gene" such as those described in the assigned Maxygen patents. The present invention is not limited to the particular methodology, protocols, vectors, and reagents, etc., described herein, as these may vary. In addition, this specification employs the terminology described above for the purpose of describing particular embodiments only and not limiting the scope of the invention. The singular forms "a", "an", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, a reference for "a gene" is a reference to one or more genes and encompasses equivalents therefore known to the skilled person, and so on.

Polynucleotide sequences The nicotinic alkaloid biosynthesis genes have been identified in several plant species, exemplified by Nicotiana plants. Accordingly, the present invention encompasses any nucleic acid molecule, gene, polynucleotide, DNA, AR, mAR, or cAR that is isolated from the genome of a plant species that regulates the biosynthesis of nicotine alkaloid. For example, the deletion of at least one of A622 and NBBl, can be used to regulate nicotine content in a plant. Accordingly, nicotinic alkaloid levels can be further reduced by suppression of a nicotine biosynthesis gene, such as at least one of QPT and PMT, and at least one of A622 and NBBl. Plants with multiple gene suppression can be obtained by plant regeneration from genetically engineered plant cells for the suppression of multiple genes or by crossing a first genetically engineered plant for suppression of a nicotine biosynthesis gene with a second plant designed genetically for suppression of at least one of A622 and NBBl. In one aspect, the invention provides an isolated nucleic acid molecule comprising SEQ ID NO: 1, polynucleotide sequences that encode a polypeptide set forth in SEQ ID NO: 2, polynucleotide sequences which hybridize to SEQ ID NO: 1 and encode an A622 polypeptide; and polynucleotide sequences which differ from SEQ ID NO: 1 due to the degeneracy of the genetic code. A peptide encoded by SEQ ID NO: 1 is a further aspect of the invention and is set forth in SEQ ID NO: 2. In another aspect, the invention provides an isolated nucleic acid molecule comprising SEQ ID NO: 3; polynucleotide sequences that encode a polypeptide set forth in SEQ ID NO: 4; polynucleotide sequences which hybridize to SEQ ID NO: 3 and encode a NBBl polypeptide; and polynucleotide sequences which differ from SEQ ID NO: 3 due to the degeneracy of the genetic code. A peptide encoded by SEQ ID NO: 3 is a further aspect of the invention and is set forth in SEQ ID NO: 4. The invention further provides nucleic acids that are complementary to SEQ ID NO: 1 or 3, additionally as a nucleic acid, comprising at least 15 continuous bases, which hybridize to SEQ ID NO: 1 or 3 under moderate or high severity conditions, as described below. For the purposes of this description, the category of nucleic acids that hybridize to SEQ ID NO: 3 is exclusive of a nucleic acid having the sequence of SEQ ID NO: 559, described in published international application WO 03/097790, and any fragment of it. In a further embodiment, a siRNA molecule of the invention comprises a polynucleotide sequence that suppresses the expression of any of SEQ ID NO 1 or 3, although the sequences set forth in SEQ ID NO: 1 or 3 are not limiting. A siRNA molecule of the invention can comprise any A622 or continuous NBB1 sequence, for example, from about 15 to about 25 or more or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , or 25 or more continuous nucleotides. In this context, too, the category of the siRNA molecules is unique to a molecule having the nucleotide sequence of the aforementioned SEQ ID NO: 559 in WO 03/097790, in addition to any fragment thereof. By "isolated" nucleic acid molecule (s) is meant a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a DNA construct are considered isolated for the purposes of the present invention. More examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or DNA molecules that are purified, partially or substantially in solution. Isolated RNA molecules include in vitro RNA transcripts of the DNA molecules of the present invention. The nucleic acid molecules isolated, in accordance with the present invention, further include such synthetically produced molecules. The nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for example, cDNA and genomic DNA obtained by cloning or synthetically produced. The DNA or AR can be concatenated or single strand. The single-stranded DNA can be the coding strand, also known as the sense strand, or it can be the uncoded strand, also called the antisense strand. Unless indicated otherwise, all nucleotide sequences determined by the sequencing of a DNA molecule herein were determined using an automated DNA sequencer (such as Model 373 from Applied Biosystems, Inc.). Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. The nucleotide sequences determined by automation are commonly at least about 95% identical, more commonly at least about 96% up to at least about 99.9% identical for the current nucleotide sequence of the sequenced DNA molecule. The current sequence can be determined more precisely by other approaches that include manual DNA sequencing methods well known in the art. As is also known in the art, a simple insertion or deletion in a given nucleotide sequence compared to the current sequence will cause a change in structure in the translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a nucleotide sequence determined can be completely different from the amino acid sequence currently encoded by the sequenced DNA molecule, which starts at the point of such an insertion or deletion. In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide that hybridizes, under severe hybridization conditions, to a portion of the polynucleotide in a nucleic acid molecule of the invention, as described above. A polynucleotide which hybridizes to a "portion" of a polynucleotide projects a polynucleotide, either DNA or RNA, which hybridizes to at least about 15 nucleotides, and more preferably to at least about 20 nucleotides, and further preferably at least about 30 nucleotides, and even more preferably more than 30 nucleotides of the reference polynucleotide. For the purpose of the invention, two sequences hybridize when they form a double stranded complex in a 6X SSC hybridization solution, 0.5% SDS, Denhardt 5X solution and 100 μg of non-specific DNA carrier. See Ausubel et al., Supra, in section 2.9, supplement 27 (1994). The sequences can hybridize to "moderate severity", which is defined as a temperature of 60 ° C in a 6X SSC hybridization solution, 0.5% SDS, 5X Denhardt's solution and 100 pg of non-specific DNA carrier. For "high severity" hybridization, the temperature is increased to 68 ° C. After the hybridization reaction of moderate severity, the nucleotides are washed in a solution of 2X SSC plus 0.05% SDS for five times at room temperature, with subsequent washes with 0. IX SSC plus 0.1% SDS at 60 ° C for 1 hr . For high severity, the wash temperature was increased to 68 ° C. For the purpose of the invention, the hybridized nucleotides are those that are detected using 1 ng of a radiolabeled probe having a specific reactivity of 10,000 cpm / ng, where the hybridized nucleotides are clearly visible after exposure to X-ray film to - 70 ° C for no more than 72 hours. The present application is directed to nucleic acid molecules which are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence described in SEQ ID NO: l or 3. Nucleic acid molecules that are at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence shown in SEQ ID NO: 1 or 3 are preferred. Differences between two nucleic acid sequences may occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or in any manner between those terminal positions, interspersed either individually between nucleotides in the reference sequence or in one or more continuous groups within the reference sequence. As a practical matter, if any particular nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to a reference nucleotide sequence it refers to a comparison made between two molecules that use algorithms standard well known in the art and can be conventionally determined using publicly available computation programs such as the BLASTN algorithm. See Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). The heterologous sequence used in the antisense methods of the present invention may be selected such as to produce a product of AR complementary to a mRNA sequence of A622 or complete NBBl, or to a portion thereof. The sequence may be complementary to any contiguous sequence of the natural messenger RNA, that is, it may be complementary to the endogenous mRNA sequence near the 5 'terminus or to the coverage site, in the 3' direction from the coverage site, between the coverage site and the initiation codon and can cover all or only a portion of the non-coding region, can bridge the non-coding region and the coding region, be complementary to all or part of the coding region, complementary to the 3 'termination of the coding region, or complementary to the 3' untranslated region of the mRNA. The appropriate antisense sequences may be from at least about 13 to about 15 nucleotides, at least about 16 to about 21 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 125 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, or more. In addition, the sequences can be extended or shortened at the 3 'and 5' endings of these. The particular antisense sequence and the length of the antisense sequence will vary, depending, for example, on the degree of inhibition desired and the stability of the antisense sequence. Generally available techniques and the information provided in this specification can guide the selection of antisense sequences A622 or NBBl. With reference to SEQ ID NO: 1 or 3 herein, an oligonucleotide of the invention can be a continuous fragment of the cDNA sequence A622 or NBBl in antisense orientation, of any length which is sufficient to achieve the desired effects when transforms into a receiving plant cell. The present invention may contemplate co-deletion of sense of one or both of A622 and NBB1. The sense polynucleotides used to carry out the present invention are of sufficient length to suppress, when expressed in a plant cell, the native expression of the plant protein A622 or NBBl in that plant cell. Such sense polynucleotides may be essentially a genomic or complementary nucleic acid encoding the A622 or NBB1 enzyme, or a fragment thereof, with fragments that are commonly at least 15 nucleotides in length. Techniques are generally available to establish the length of sense DNA that results in suppression of the expression of a native gene in a cell. In an alternate embodiment of the present invention, plant cells are transformed with a nucleic acid construct that contains a segment of the polynucleotide, which encodes an enzyme RNA molecule (a "ribozyme"), whose enzyme RNA molecule is directed against (ie, unfolds) the mRNA transcript of DNA encoding A622 or NBB1, as described herein. Ribozymes contain domains that bind to the substrate, which bind to the accessible regions of the target mRNA, and domains that catalyze the cleavage of RNA, preventing the translation and production of the protein. The binding domains may comprise antisense sequences complementary to the target mRNA sequence; the catalytic portion may be a hammerhead portion or other portions, such as the fork-like portion. The cleavage sites of the ribozyme within a target RNA can be identified initially by examining the target molecule for 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 for structural features can be evaluated. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with the complementary oligonucleotides, using the ribonuclease protection assays as are known in the art. The DNA encoding enzymatic RNA molecules can be produced according to known techniques. For example, see Cecil et al, U.S. Pat. No. 4,987,071; Keene et al, U.S. Patent No. 5,559,021; Donson et al, U.S. Patent No. 5,589,367; Torrence et al, U.S. Patent No. 5,583,032; Joyce, U.S. Patent No. 5,580,967; Gold et al., U.S. Patent No. 5,595,877; Wagner et al, U.S. Patent No. 5,591,601; and the U.S. patent No. 5,622,854.

The production of such an enzyme RNA molecule in a plant cell and disruption of the production of the protein A622 or NBBl reduces the activity of the protein in the cells of the plant, in essentially the same way as the production of a molecule of antisense RNA; that is, interrupting the translation of the mRNA in the cell that produces the enzyme. The term "ribozyme" describes a nucleic acid that contains RNA, which functions as an enzyme, such as an endoribonuclease, and can be used interchangeably with the "enzyme RNA molecule". The present invention further includes, nucleic acids encoding ribozymes, nucleic acids encoding ribozymes and having been inserted into an expression vector, host cells containing such vectors, and methodology employing ribozymes to decrease A622 expression. and NBBl in plants. In one embodiment, the present invention provides double-stranded nucleic acid molecules that mediate silencing of the RNA interference gene. In another embodiment, the siNA molecules of the invention consist of double nucleic acid molecules containing from about 15 to about 30 base pairs between oligonucleotides comprising from about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In another embodiment, the siNA molecules of the invention comprise double nucleic acid molecules with pendant ends of from about 1 to about 32 (eg, about 1, 2, or 3) nucleotides, eg, about 21 double nucleotides with about 19 pairs base and a 3 'terminal mononucleotide, dinucleotide, or trinucleotide projections. In yet another embodiment, the siNA molecules of the invention comprise double nucleic acid molecules with blunt-ended ends where both ends are blunt-ended, or alternatively, where one end is blunt-ended. A siNA molecule of the present invention can comprise modified nucleotides while maintaining the ability to mediate RNAi. Modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, and / or bioavailability. For example, a siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. As such, a siNA molecule of the invention can generally comprise from about 5% to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides).

The actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single stranded, the percentage change can be based on the total number of nucleotides present in the single stranded siNA molecules. Likewise, if the siNA molecule is double-stranded, the modification in percent can be based on the total number of nucleotides present in the sense strand, antisense strand, or both, in the sense and antisense strand. For example, the expression A622 and NBB1 can be decreased through genetic engineering methods that are well known in the art. Expression can be reduced by introducing a nucleic acid construct that results in the expression of an RNA, comprising a portion of a sequence encoding A622 or NBBl. The portion of the sequence may be in the sense sense or antisense. The portion of the sequence may be present in inverted repeats capable of forming a double-stranded RNA region. Expression can be reduced by introducing a nucleic acid construct encoding an enzyme RNA molecule (i.e., a "ribozyme"), whose enzyme RNA molecule is directed against (ie, unfolds) the niRNA DNA transcript encoding A622 or NBBl. Expression can be reduced by introducing a nucleic acid comprising a portion of an A622 or NBB1 sequence that causes directed in situ mutagenesis of an endogenous gene, causing its inactivation.

Sequence analysis Sequence alignment methods for comparison are well known in the art. Optimal alignment of sequences for comparison can be addressed by means of the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by searching for the similarity method of Pearson and Lipman, Proc. Nati Acad. Sci. USA 85: 2444 (1988); through computerized applications of these algorithms, which include, but are not limited to: CLUSTAL in the PC / Gene program of Intelligenetics, Mountain View, California; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet et al, Nucleic Acids Research, 16: 10881-90 (1988); Huang et al, Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson et al, Methods in Molecular Biology 24: 307-331 (1994).

The BLAST family of programs that can be used for similarity searches in the database includes: BLASTN for the sequences that trace the nucleotide against the nucleotide database sequences; BLASTX for the sequences that trace the nucleotide against the sequences in the protein database; BLASTP for sequences that track the protein against the protein's database sequences; TBLASTN for the sequences that trace the protein against the sequences in the nucleotide database; and TBLASTX for the sequences that trace the nucleotide against the sequences in the nucleotide database. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel et ah, Eds. , Greene Publishing and Wiley-Interscience, New York (1995); Altschul et al, J. Mol. Biol. 215: 403-410 (1990); and Altschul et al, Nucleic Acids Res. 25: 3389-3402 (1997). Software to perform BLAST analyzes is publicly available, for example, through the National Center for Biotechnology Information. This algorithm involves first identifying high qualification sequence pairs (HSPs) by identifying short words of length W in the tracking sequence, either matching or satisfying some T record in the positive value threshold when aligning with a word of the same length in a sequence of the database. The T refers to the registration of the immediate word in the threshold. These initial immediate word hits act as seeds to initiate searches and find larger HSPs that contain them. The word hits then extend in both directions along each sequence such that the cumulative alignment record can be increased. Cumulative records are calculated using, for the nucleotide sequences, parameters M (record awarded for a pair of matching residues, always> 0) and N (record of penalty for matching residues, always <0). For amino acid sequences, a register matrix is used to calculate the cumulative record. The extension of word hits in each direction stops when: the cumulative alignment record falls by the amount X of its maximum value obtained; the cumulative record goes from zero to below, due to the accumulation of one or more alignments of the negative record residue; or at the end of any sequence that is reached. The parameters of the BLAST algorithm W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11 as a default, a probability (E) of 10, a cut of 100, M = 5, N = -4, and a comparison of both strands. For the amino acid sequences, the BLASTP program uses a word length (W) of 3, a probability (E) of 10, and the register matrix BLOSUM62 as default values. See Henikoff &; Henikoff, Proc. Nati Acad. USA 89: 10915 (1998). In addition to calculating the percent identity of the sequence, the BLAST algorithm also performs a statistical analysis of the similarity between the two sequences (see, for example, Karlin &Altschul, Proc. Nati, Acad. Sci. USA, 90: 5873 -5877 (1993)). A measure of similarity provided by the BLAST algorithm is the probability of the smallest sum (P (N)) that provides an indication of the probability by which the coincidence between two nucleotide or amino acid sequences would occur by chance. The multiple alignment of the sequences can be performed using the CLUSTAL alignment method (Higgins &Sahrp, CABIOS 5: 151-153 (1989)) with the predefined parameters (INTERVAL PENALTY = 10, INTERVAL PENALTY LENGTH = 10). The predefined parameters for the alignments in pairs using the CLUSTAL method are KTUPLE 1, PENALTY OF INTERVAL = 3, WINDOW = 5 and DIAGONALS SAVED = 5. The following parameters for runs are preferred to determine the alignments and similarities using BLAST that contributes to the E values and percentage of identity for the polynucleotide sequences: Unix run command: blastall-p blastn -d embldb -e 10 - GO - EO - rl - v30 - b 30-queryseq i - or results; the parameters are: -p Program name [Cord]; - Database [Cordon]; - Expectation value (E) [Real]; - cost G to open an interval (zero invokes the predefined behavior) [Integer]; - E Cost to extend an interval (zero invokes the predefined behavior) [Integer]; - r prize for a nucleotide match (only blastn) [Integer]; - v Number of online descriptions (V) [Integer]; - b Number of alignments to show (B) [Integer]; - i Trace file [File In]; and - o Output file for the BLAST report [File Outside] Optional. The "Successes" to one or more sequences of the database through a sequence in question produced by BLASTN, FASTA, BLASTP or a similar algorithm, aligns and identifies similar portions of sequences. The hits are placed in the order of the degree of similarity and the overlap length of the sequence. The hits for a database sequence generally represent an overlap on only a fraction of the length of the sequence of the sequence in question. The BLASTN, FASTA and BLASTP algorithms also produce "expected" values for the alignments. The expected value (E) indicates the number of hits one can "expect" to see over a certain number of immediate sequences by chance when investigating a database of a certain size. The expected value is used as an important threshold to determine whether the success for a database, such as the preferred EMBL database, indicates true similarity. For example, an E value of 0.1 assigned to a hit of the polynucleotide is interpreted as being that in a database of the size of the EMBL database, one might expect to simply see by chance 0.1 matches over the aligned portion of the sequence with similar record. By this criterion, the aligned and tied portions of the polynucleotide sequences have a 90% probability of being the same. For sequences that have an E value of 0.01 or less over the aligned and tied portions, the probability of finding a coincidence by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm. According to one embodiment, the "variant" polynucleotides, with respect to each of the polynucleotides of the present invention, preferably comprise sequences having the same or fewer nucleic acids in which each of the polynucleotides of the present invention they produce an E-value of 0.01 or less when compared to the polynucleotide of the present invention. That is, a variant polynucleotide is any sequence that has at least a 99% chance, which is equal to that of the polynucleotide of the present invention, as measured having an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms establish the parameters described above. Alternatively, the variant polynucleotides of the present invention hybridize to the polynucleotide sequence: 1 or 3, or complements, reverse sequences, or inverse complements of those sequences, under severe conditions. The present invention also encompasses polynucleotides which differ from the described sequences but which, as a consequence of the degeneracy of the genetic code, encode a polypeptide which is the same as that encoded by a polynucleotide of the present invention. Thus, polynucleotides comprising sequences that differ from the polynucleotide sequences described in SEQ ID NO: 3 or complements, reverse sequences, or inverse complements thereof, as a result of the conservative substitutions contemplated by and encompassed within the present invention. Additionally, polynucleotides comprising sequences that differ from the polynucleotide sequences described in SEQ ID NO: 1 or 3, or complements, inverse complements or inverse sequences thereof, as a result of deletions and / or insertions of a total of less than 10% of the total sequence length are also contemplated by and encompassed within the present invention.

In addition to having a specified percent identity for an inventive polynucleotide sequence, the variant polynucleotides preferably have an additional structure and / or functional characteristics in common with the inventive polynucleotide. In addition to sharing a high degree of similarity in their primary structure in the polynucleotides of the present invention, polynucleotides having a degree of identity specific to or capable of hybridizing to an inventive polynucleotide preferably have at least one of the following characteristics: (i) contain an open reading structure or partial open reading frame that encodes a polypeptide having substantially the same functional properties as those of the polypeptide encoded by the inventive polynucleotide; or (ii) they have domains in common. For example, a variant polynucleotide can encode a polypeptide that has the ability to suppress A622 or NBB1.

Nucleic Acid Constructs According to one aspect of the invention, a sequence that reduces nicotinic alkaloid biosynthesis is incorporated into a nucleic acid construct that is convenient for plant transformation. For example, such a nucleic acid construct can be used to decrease at least one A622 or NBB1 expression gene in plants. Additionally, an inventive nucleic acid construct can decrease one or both of the A622 and NBB1 expressions, as well as a polynucleotide sequence encoding an enzyme from the biosynthesis of nicotine. Accordingly, nucleic acid constructs are provided which comprise a sequence that down-regulates nicotinic alkaloid biosynthesis, under the control of a transcriptional initiation region operative in a plant, so that the construct can generate RNA in a host cell of the plant. Recombinant DNA constructs can be made using standard techniques. For example, the DNA sequence for transcription can be obtained by treating a vector containing said sequence with restriction enzymes to cut the appropriate segment. The DNA sequence for transcription can also be generated by combining basic pairs and linking synthetic oligonucleotides or using synthetic oligonucleotides in a polymerase chain reaction (PCR) to provide the appropriate restriction sites for each purpose. The DNA sequence is then cloned into a vector containing suitable regulatory elements, such as the upstream promoter and downstream terminator sequences.

Appropriate Regulatory Elements The promoter connotes a DNA region upstream at the start of transcription, which is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A "constitutive promoter" is one that is active throughout the life of the plant and under most environmental conditions. The specific tissue, preferred tissue, specific cell type and inducible promoters constitute the class of "non-constitutive promoters". "Operably linked" refers to a functional link between a promoter and a second sequence, wherein the promoter sequence initiates and mediates the transcription of the DNA sequence corresponding to the second sequence. In general, "operably linked" means that the nucleic acid sequences that are linked are immediate. Promoters useful for the expression of a nucleic acid sequence introduced into a cell to reduce the expression of A 622 or NBBl can be constitutive, or tissue-specific, preferred tissue, specific cell type, and inducible promoters. Preferred promoters include promoters that are active in root tissues, such as tobacco RB7promoter (Hsu et al Pestic Sci 44: 9-19 (1995), US Patent No. 5,459,252) and promoters that are activated under conditions which produce a high expression of enzymes involved in nicotine biosynthesis such as the tobacco RD2 promoter (US Patent No. 5,837,876), PMT promoters (Shoji T. et al, Plant Cell Physiol, 41: 831-839 (2000b); WO 2002/038588) or a 4622 promoter (Shoji T. et al, Plant Mol Biol, 50: 427-440 (2002)). The vectors of the invention may also contain termination sequences, which are positioned downstream of the nucleic acid molecules of the. invention, such transcription of mRNA is terminated, and the pulley sequences are added. Examples of such terminators are the 35S terminator of cauliflower mosaic virus (CaMV) and the nopaline synthase gene terminator (Tnos). The expression vector may also contain enhancers, start codons, splice signal sequences, and targeting sequences. The expression vectors of the invention may also contain a selection marker by which the transformed plant cells can be identified in the culture. The label can be associated with the heterologous nucleic acid molecule, i.e., the gene is operably linked to a promoter. As used herein, the term "marker" refers to a gene that encodes a characteristic or phenotype that allows the selection of, or separation by exclusion for a plant or cell of the plant containing the marker. Normally, the marker gene will code the antibiotic or herbicide resistance. This allows the selection of transformed cells from cells that do not transform or transfected. Examples of suitable selectable markers include adenosine deaminase, dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidine kinase, xanthine-guanine phospho-ribosyltransferase, glyphosate and glufosinate resistance, and amino-glycoside 3'-0-phosphotranserase (kanamycin, neomycin and resistance G418). These markers include resistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin. The construct may also contain the selectable marker Bar gene that confers resistance to the herbicidal phosphinothricin analogues such as glufosinate-ammonium. The Thompson et al., EMBO J. 9: 2519-2523 (1987). Other suitable selection markers are also known. Visible markers such as green fluorescent protein (GFP) can be used. Methods for identifying or selecting transformed plants based on the control of cell division have also been described. See WO 2000/052168 and WO 2001/059086. Replication sequences, of bacterial or viral origin, may also be included to allow the vector to be cloned into a phage or bacterial host. Preferably, a broad host range of prokaryotic origin is used for replication. A selectable marker for the bacteria can be included to allow the selection of bacterial cells carrying the desired construct. Suitable prokaryotic selectable markers also include resistance to antibiotics such as kanamycin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art. For example, when Agrobacteriuin is the host, T-DNA sequences can be included to facilitate subsequent transfer and incorporation into plant chromosomes.

Plants for Genetic Engineering The present invention comprises the genetic manipulation of a plant to suppress nicotinic alkaloid synthesis via the introduction of a polynucleotide sequence that downregulates the expression of a gene, such as A622 and NBB1, which regulate nicotinic alkaloid synthesis. The result is a plant that has reduced nicotinic alkaloid levels. In this description, "plant" denotes any nicotinic alkaloid containing plant material that can be genetically engineered, including but not limited to differentiated or undifferentiated plant cells, protosomes, whole plants, plant tissues, or organs of the plant, or any component of a plant such as a leaf, stem, root, shoot, tuber, fruit, rhizome, or the like. Illustrative plants that can be designed in accordance with the invention include but are not limited to tobacco, potato, tomato, egg plant, green pepper, and Atropa belladonna.

Transformation and Plant Selection The constructs according to the invention can be used to transform any cell of the plant, using a suitable transformation technique. Both cells of angiosperm and dicotyledonous and monocotyledonous gymnosperms can be transformed in various ways known in the art. For example, see Klein et al, Biotechnology 4: 583-590 (1993); Bechtold et al, C, R. Acad. Sci Paris 316: 1194-1199 (1993); Ben et al, Mol. Gen. Genet. 204: 383-396 (1986); Paszowski ah, EMBO J. 3: 2717-2722 (1984); Sagi et al, Plant Cell Rep. 13: 262-266 (1994). Agrobacterium species such as A. tumefaciens and A. rhizogenes can be used, for example, according to Nagel et al, Microbiol Lett 61: 325 (1990). Additionally, plants can be transformed by Rhizobium, Sinorhizobium or Mesorhizobium transformation. Broothaerts et al, Nature 433: 629-633 (2005).

For example, the Agrobacterium can be transformed with an expression vector of the plant via, for example, electroporation after the Agrobacterium is introduced into the plant via the cells, for example, the well-known leaf-disk method. Additional methods to accomplish this include, but are not limited to, electroporation, particle bombardment, calcium phosphate precipitation, and polyethylene glycol fusion, transfer of pollen grains on germination, direct transformation (Lorz et al, Mol. Genet, 199: 179-182 (1985)), and other methods known in the art. If a selection marker, such as kanamycin resistance, is used, it becomes easier to determine which cells have been transformed successfully. The transformation methods of the Agrobacterium discussed above are known to be useful for transforming dicots. Additionally, de la Peña et al, Nature 325: 274-276 (1987), Rhodes et al, Science 240: 204-207 (1988), and Shimamato et al, Nature 328: 274-276 (1989) has transformed the monocotyledons of the cereal using Agrobac erium. Also see Bechtold et al., CR. Acad. Sci. Paris 316 (1994), which illustrates vacuum infiltration for transformation mediated by Agrobacterium. For the purposes of this description, a plant or cell of the plant can be transformed with a plasmid comprising one or more sequences, each operably linked to a promoter. For example, an illustrative vector may comprise a QPT sequence operably linked to a promoter. Similarly, the plasmid can comprise a QPT sequence operably linked to a promoter and an A622 sequence operably linked to a promoter. Alternatively, a plant or cell of the plant can be transformed with more than one plasmid. For example, a plant or cell of the plant can be transformed with a first plasmid comprising a QPT sequence operably linked to a promoter, which is different from a second plasmid comprising an A622 or NBBl sequence. Of course, the first and second plasmids or portions thereof are introduced into the same cell of the plant. Genetically, the engineered plants of the invention can be produced by conventional breeding. For example, a genetically engineered plant having sub-QPT and A622 activity can be produced by crossing a transgenic plant having reduced QPT expression with a transgenic plant having reduced A622 expression. Following the successive rounds of crossing and selection, a genetically engineered plant that has sub-QPT and A622 activity can be selected. The presence of a protein, polypeptide, or nucleic acid molecule in a particular cell can be measured to determine whether, for example, a cell has been transformed or transfected successfully. Marker genes can be included within pairs of recombination sites recognized by specific recombinases such as ere or flp to facilitate marker removal after selection. See the published application U. S. No. 2004/0143874. Transgenic plants without the marker genes can be produced using a second plasmid comprising a nucleic acid encoding the marker, other than a first plasmid comprising an A622 or NBBl sequence. The first and second plasmids or portions thereof are introduced into the same cell of the plant, such that the selectable marker gene that is temporarily expressed, the cells of the transformed plant are identified, and transformed plants are obtained in which the A622 or NBBl sequence is stably integrated into the genome and the selectable marker gene is not stably integrated. See published application U. S. No. 2003/0221213. The first plasmid comprising an A622 or NBB1 sequence can optionally be a binary vector with a T-DNA region that is completely made up of nucleic acid sequences present in the non-transgenic N. tabacum of wild type or sexually compatible Nicotiana species. The cells of the plant can be transformed with the nucleic acid constructs of the present invention without the use of a selectable marker or visible marker and the tissue of the transgenic plant and transgenic regenerated plants can be identified by detecting the presence of the construct introduced by PCR or other detection methods for specific nucleic acid sequences. The identification of cells of transformed plants can be facilitated by the recognition of differences in the growth rate or a morphological characteristic of said cell of the transformed plant compared to the growth rate or morphological characteristic of a cell of the non-transformed plant that is grown under similar conditions (see WO 2004/076625). The methods for regenerating a transgenic plant of a transformed cell or culture vary according to the species of the plant but are based on the known methodology. For example, methods for regenerating transgenic tobacco plants are well known. For the purposes of the present disclosure, genetically engineered plants having sub-expression of at least one of A622 and NBB1 are selected. Additionally, inventive genetically engineered plants may have downregulated expression of a nicotine biosynthesis gene, such as QPT or PMT, and at least one of A622 and NBBl.

Nicotine serves as a natural pesticide that helps protect tobacco plants from pest damage, an increase in the susceptibility to conventional breeding of low transgenic nicotine tobacco due to insect damage has been reported. Legg, P.D., et al, Can J. Cyto., 13: 287-291 (1971); Voelckel, C, et al, Chemoecology 11: 121-126 (2001); Steppuhn, A., et al, PLoS Biol, 2 (8): e217: 1074-1080 (2004). Accordingly, it may be further desirable to transform nicotine-reduced plants produced by the methods present with a transgene that will confer additional protection to insects, such as the gene encoding an insecticidal Bt protein, proteinase inhibitor, or protein binding. to biotin. A transgene that confers additional insect protection can be introduced by crossing a transgenic nicotine-reduced plant with a second transgenic plant that contains a gene that codes for a protein with insect resistance.

Quantification of the nicotinic alkaloid content The transgenic plants of the invention are characterized by their diminished nicotinic alkaloid content. The diminished nicotinic alkaloid content in the genetically engineered plant is preferably achieved via the decreased expression of a gene with a path towards nicotine biosynthesis, such as A622 or NBBl. In describing a plant of the invention, the phrase "nicotinic or reduced nicotine alkaloid content" refers to a quantitative reduction in the amount of nicotinic alkaloid in the plant when compared to an untransformed control plant. A quantitative decrease in nicotinic alkaloid levels can be tested by several methods, such as by quantification based on liquid gas chromatography, high performance liquid chromatography, radioimmunoassays, and enzyme-linked immunosorbent assays. In the present invention, nicotinic alkaloid levels were measured by liquid gas chromatography equipped with a capillary column and an FID detector, as described in Hibi, N. , et al, Plant Physiology 100: 826-835 (1992).

Reduced Products in nicotinic alkaloids The present invention provides a transgenic plant having reduced levels in nicotinic alkaloids. For example, the present invention contemplates reducing nicotine levels by suppressing at least one expression of A622 and NBBl. Following the selection of a transgenic plant having deleted A622 or NBBL expression and reduced nicotine content, a variety of products of such a plant can be made.

Because the invention provides a method for reducing alkaloids, TSNAs can also be reduced because there is a significant positive correlation between the alkaloid content in tobacco and the accumulation of TSNA. For example, a significant correlation coefficient between anatabine and NAT was 0.76. Djordjevic et al, J. Agrie. Food Chem., 37: 752-756 (1989). TSNAs are a class of carcinogens that are predominantly formed in tobacco during curing, processing and smoking. However, TSNAs are present in small amounts in growing tobacco plants or fresh cut tobacco. Hecht & Hoffman, J., Nati. Cancer Inst. 58, 1841-4 (1977); Wiernik et al, Recent Adv. Tob. Sci, 21: 39-80 (1995). The nitrosamines, which contain the organic functional group, N-N = 0, are formed by the easy addition of a group N = 0 by an agent having nitrogen to a nitrogen of a secondary or tertiary amine. This particular class of carcinogens is only found in tobacco although they could potentially occur in other products that contain nicotine. The TSNAs are considered among the most prominent carcinogens in cigarette smoke and their carcinogenic properties are well documented. See Hecht, S., Mutat. Res. 424: 127-42 (1999); Hecht, S., Toxicol. 11, 559-603 (1998); Hecht, S., et al., Cancer Surv. 8, 273-294 (1989). TSNAs have been cited as the causes of oral cancer, esophageal cancer, pancreatic cancer, and lung cancer (Hecht &Hoffman, IARC Sci. PuU, 54-61 (1991)). In particular, TSNAs are implicated as the causative agent in the dramatic emergence of adenocarcinoma associated with cigarette smoking and lung cancer (Hoffmann et al, Crit. Rev. Toxicol 26, 199-211 (1996)). The four TSNAs are considered the most important for their levels of exposure and carcinogenic potency and have been reported to be possibly carcinogenic to humans is -nitrosonornicotine (NNN), 4-methylnitrosamino-l- (3-pyridyl) -1 -butanone (NNK), '-nitrosoanatabine (NAT) and' -nitrosoaiiabasina (NAB) Reviewing in XA C monographs on the evaluation of the carcinogenic risk of chemical to humans. Lyon (France) Vol 37, pp.205-208 (1985). These TSNAs are formed by nicotine N-nitrosation and minor Nicotiana alkaloids that include nornicotine, anatabine and anabasine. The following levels of alkaloid compounds have been reported for the main stream smoke of unfiltered cigars (measured in ug / cigar): nicotine: 100-3000, nornicotine: 5-150, anatabine: 5-15, Anabasine: 5- 12 (Hoffmann et al, Chem. Res, Toxicol 14: 7: 767-790 (2000)). Smoke from the main stream of American cigars, with or without filter tips, contains (measured in ng / cigar): 9-180ng NNK, 50-500ng NNN, 3-25ng NAB and 55-300ng NAT. Hoffmann, et al., J. Toxicol. Environ. Health 41: 1-52 (1994). It is important to note that the levels of these TSNAs in the smoke of the lateral flow are 5-10 times above those fumes of the main flow. Hoffmann, et al (1994). Xie et al. (2004) reported that Vector 21-41, which is a nicotine-reduced GE tobacco due to the under-regulation of QPT, has a total alkaloid level of approximately 2300 ppm, which is less than 10 percent of tobacco. wild type. Smoke from the main stream of the Vector of Cigars 21-41 had less than 10 percent NNN, NAT, PRENDE, and NNK compared to such levels of a standard full flavor cigar produced by the wild type tobacco. The strategy to reduce TSNAs by reducing the corresponding tobacco alkaloid precursors is currently the main focus of agricultural tobacco research. Siminszky et al., Proc. Nat. Acad. Sci. USA 102 (41) 14919-14924 (2005). Thus, to reduce the formation of all TSNAs there is an urgent need to reduce the precursor nicotinic alkaloids as much as possible for genetic engineering. Among others, U.S. Nos. 5,803,081, 6,135,121, 6,805,134, 6,907,887 and 6,959,712 and the U.S. Published Application. Nos. 2005/0034365 and 2005/0072047 discuss methods to reduce tobacco-specific nitrosamines (TSNAs).

A nicotine-reduced tobacco product can be in the form of tobacco leaf, tobacco made of strips, cut tobacco and tobacco fragments. A nicotine-reduced tobacco product may include cigar tobacco, cigar tobacco, tobacco powder, chewing tobacco, pipe tobacco, and cigars made from nicotine-reduced GE tobacco for use in smoking cessation therapies. Nicotine-reduced tobacco can also be used to produce reconstituted tobacco (Recon). Recon is produced from tobacco stems and / or smaller leaf particles by a process that closely resembles typical papermaking. This process brings with it the process of several portions of tobacco that will be made in Recon and cut of the tobacco into a size and shape that resemble a brownish cut made from the whole leaf of the tobacco. This cut is then mixed with the brown cut of the tobacco and prepared for the production of the cigar.

In addition to traditional tobacco products, such as cigars and pure tobacco, nicotine-reduced tobacco can be used as a source of protein, fiber, ethanol, and animal feed. See Application U.S. Published No. 2002/0197688. For example, nicotine reduced tobacco can be used as a source of Rubisco (ribose bisphosphate carboxylase-oxygenase or fraction 1 protein) because other different plants, Rubisco derived from tobacco can be easily extracted in crystalline form. With the exception of slightly lower levels of methionine, the Rubisco content of essential amino acids equals or exceeds the FAO Provisional Standard. Ershoff, B.H., et al. Society for Experimental Biology and Medicine 157: 626-630 (1978); Wildman, S.G. Photosynthesis Research 73: 243-250 (2002)). For biocombus tibies, replacing a regular portion of non-renewable or world-dependent energy sources, co-products such as Rubisco, is required to help defray the cost of producing this renewable energy. Greene et al. , Growing Energy. How Biofuels Can End America 's Oil Dependence; National Resources Defense Counsel (2004). Thus, a greater reduction of nicotinic alkaloids in tobacco creates a high probability of having a successful biomass system in tobacco. Specific examples of methods for identifying sequences encoding the enzymes involved in nicotine are presented below, as well as for introducing the target gene to produce the transformants of the plant. These are intended to exemplify and not limit the present invention.

Example 1: Preparation of the pARNi-A622 vector to reduce the alkaloid content by the sub-A622 expression The plasmid pHANNlBAL, see Wesley et al, Plant J. 27: 581-590 (2001), was modified to produce the plasmid pHANNIBAL-X as is shown in Figure 2. A Sad restriction site between the ampicillin resistance (Amp) and the 35S promoter was removed by a Sad cut and with blunt tips of subsequent DNA and ligation. The multiple cloning sites (MCS) were modified as follows. A Bam H I restriction site was added to the MCS between the promoter and the Pdk intron by inserting an adapter (5 'TCGAACGGGATCCCGCCGCTCGAGCGG) between the Xhol and EcoRI sites. A Bam Hl site was removed from a Sac I site and inserted into the MCS between the intron and the terminator by inserting an adapter (5 'GATCAGCTCTAGAGCCGAGCTCGC) between the BamHI and Xbal sites. A binary vector of the RNAi plant was prepared using pHANNIBAL-X by using a schematic diagram of Figure 3, where the different "sense" and "antisense" fragments are first obtained by the addition of specific restriction sites to the ends of a segment of the gene of interest, and then the sense and antisense fragments are inserted into the desired orientations in the modified plasmid pHANNIBAL-X.

The DNA segment containing the sense and antisense fragments and the intervention intron Pdk was replaced for the GUS encoding the pBI121 region (Wesley et al. to produce a binary RNAi vector. The 814 bp-1160 bp region of the A622 cDNA was used as the region that forms the dsRNA (sense chain, antisense chain). PCR was performed using A622 cDNA cloned in the pcDNAII as the template and the primers with the additional bases encoding the indicated restriction enzyme sites and the target DNA fragment was collected and the TA was cloned for a pGEM-vector. t. The sequences of the primers used were: The sense strand A622 F814-Xhol-A622 Rl 160- A622 F814-Xhol 5 'CCGCTCGAGCGGTCAGAGGAAGATATTCTCCA 3' A622 Rll60- 5 'GGGGTACCCCTGGAATAAGACGAAAAATAG_3_' Antisense Chain A622 F814-Xbal - A622 Rll60-Clal A622 F814-Xbal 5 'GCTCTAGAGCTCAGAGGAAGATATTCTCCA 3' A622 RI 1 60-ClaI 5 'CCATCGATGGTGGAATAAGACGAAAAATAG_3_' Recombination with the modified pHANNIBAL-X was performed by initiating with the sense strand followed by the antisense strand. The cloned DNA fragments TA were cut with the appropriate restriction enzymes, pooled and ligated to pHANNIBAL-X which was cut with the same restriction enzymes. The resulting plasmid contains a DNA sequence with inverted repeats of fragment A622 separated by intron Pdk. The R Ai region was cut from pHANNIBAL-X with the sense and antisense strands incorporated by treating them with BamH I and Sac I and ligated to pBI121 from the GUS coding region that had been removed by the similar treatment to produce the vector binary pRNAi-A622 for the transformation of the plant containing a segment of T-DNA (Figure 4) containing a selectable marker cassette nptll and the cassette of RNAi A622.

Example 2: Suppression of A622 in tobacco BY-2 cells. Although the BY-2 tobacco cell cultures did not synthesize nicotinic alkaloids, the treatment of methyl jasmonate induces the expression of genes for known enzymes in the path of nicotine biosynthesis and extracts the formation of nicotinic alkaloids. To deduce the function of A622, cells cultured with the RNAi strain were prepared in which the mRNA of pRNAi-A622 was expressed in BY-2 cells of tobacco grown to suppress A622. Agrotrans formation The vector (pRNAi-A622) was transformed into the EH105 strain Agrobacterium turnefaciens which was used to transform the tobacco BY-2 cells. The methods for infecting and selecting tobacco BY-2 cells are as follows. Four mi of BY-2 cells that had been cultured for 7 days in 100 ml of the modified LS medium, see Imanishi et al, Planta Mol. Biol., 38: 1101-1111 (1998), were subcultured in 100 ml modified with the LS medium, and cultured for 4 days. One hundred microliters of the solution of A. tumefaciens that had been cultured for 1 day in the YEB medium was added to the 4 ml of cells that had been cultured for 4 days, and the two were cultured at the same time for 40 hours in the dark. at 27 ° C. After the culture, the cells were washed twice with the modified LS medium to remove the Agrobacteria. The washed cells were propagated in a modified LS selection medium containing kanamycin (50 mg / 1) and carbenicillin (250 mg / 1), and the transformed cells were selected. After being cultured for approximately 2 weeks in the dark at 27 ° C, the transformed cells were transferred to a fresh modified LS selection medium, and cultured in the dark for 1 week at 27 ° C. The transformed cells were then grown in a suspension culture in the dark at 27 ° C for 1 week in 30 ml of modified liquid LS medium. 1 ml of the cultured transformed cells were subcultured in 100 ml of the modified LS medium. Transformed cells were subcultured every 7 days in the same manner as wild-type cells.

Synthesis of 10 ml alkaloids from each of the transformed BY-2 cells that had been cultured for 7 days and the cultured tobacco cells that had been transformed using a green fluorescent protein (GFP) expression vector as control, was washed twice with the modified LS medium that did not contain 2,4-D, after the addition of the modified LS medium that did not contain 2,4-D for a total of 100 ml, were cultured in a suspension at 27 ° C for 12 hours. hours. After the addition of 100 μ? of methyl asmonate (MeJa) which had been diluted to 50 μ? with DMSO, the cells of the suspension were cultured for 48 hours at 27 ° C. The cells treated with the Jasmonate were filtered, pooled and dehydrated by freezing. The sulfuric acid, 3 ml of 0.1 N, was added to 50 mg of the sample dehydrated by freezing. The mixture was sonic for 15 minutes, and filtered. A solution of 28% ammonium was added to 1 ml of the filtrate, and centrifuged for 10 minutes at 15,000 r.p.m.

One mi of the supernatant was added to an Extrelut-1 column (Merck) and allowed to settle for 5 minutes. This was eluted with 6 ml of chloroform. The eluate was then dried under reduced pressure at 37 ° C with an evaporator (Taitec Concentrator TC-8). The dry sample was dissolved in 50 μ? of ethanol solution containing 0.1% dodecane. Gas chromatography (GC-14B) provided with a capillary column was used and an FID detector was used to analyze the samples. A column of Amina RESTEC Rtx-5 (Restec) was used as a capillary column. The temperature of the column was maintained at 100 ° C for 10 min, elevated at 150 ° C at 25 ° C / min, maintained at 150 ° C for 1 min, elevated at 170 ° C at ° C / min, maintained at 170 ° C for 2 min, elevated at 300 ° C at 30 ° C / min, and then kept at 300 ° C for 10 min. The injection and detection temperature were 300 ° C. A μ? of each sample was injected and the nicotinic alkaloids were quantified by the internal standard method. As shown in Figure 5, in the BY-2 transgenic lines in which the expression of the A622 gene was suppressed by R Ai (A3 lines, A21 A33 and A43), the extraction of the jasmonate did not produce high accumulation of anatabine (alkaloid higher in the extracted cultured cells), anatalin, nicotine, or anabasine, compared to the control cell lines.

Expression of RNA To determine whether the reduction in alkaloid accumulation in the A622-K AI lines is specifically related to the reduction of A622 expression, rather than an indirect effect on the levels, of gene expression for the enzymes known in the Nicotine biosynthesis trajectory, A 622 expression levels and other genes were measured in lines treated with methyl jasmonate, transgenic lines and control lines. The total RNAs were isolated from the transgenic and wild type BY-2 cell lines which were treated with 50 μ? MeJA for 48h. The RNA levels of specific genes were determined by RT-PCR. The RNA was extracted using a RNeasy Plant mini kit (Qiagen) in accordance with the manufacturer's instructions. The cDNA was synthesized using random exometers and SuperScript first-strand synthesis system RT-PCR (Invitrogen). RT-PCR was carried out with 5 ng of the cDNA as a model using a TaKaRa ExTaq (Takara Bio) under the following conditions: for the detection of A622, NBB1, AO, QS, QPT, ODC, 22 94 ° cycles C for 30 sec, 57 ° C for 30 sec, and 72 ° C for 30 sec, for the detection of PMT, 24 cycles of 94 ° C for 1 min, 52 ° C for 30 sec, and 72 ° C for 1 min .

A622 primers: A622-07F 5 'ATGGTTGTATCAGAGAAAAG A622-05R 5' CCTTCTGCCTCTATCATCCTCCTG Primers NBB1: NBB1-01F 5 'ATGTTTCCGCTC ATAATTCTG NBBB1-1365 5' TCTTCGCCCATGGCTTTTCGGTCT AO primers: AO RT-1 5 'CAAAACCAGATCGCTTGGTC AO RT-25' CACAGCACTTACACCACCTT QS primers: QS RT-1 5"CGGTGGAGCAAAAGTAAGTG QS RT-2 5 'GAAACGGAACAATCAAAGCA QPT primers: QPT RT-1 5 'TCACTGCTACAGTGCATCCT QPT RT-2 5' TTAGAGCTTTGCCGACACCT ODC primers: ODC RT-1 5 'CGTCTCATTCCACATCGGTAGC ODC RT-2 5' GGTGAGTAACAATGGCGGAAGT Primers P: PMT RT-1 5 GCCATGATAATGGCAACGAG PMT RT-2 5 TTAGCAGCGAGATAAGGGAA As shown in Figure 6, the A622 is not induced in the silenced lines A622. Other genes for the enzymes of the known nicotine biosynthetic pathway are induced. These results provide evidence that A622 is included in the path of nicotinic alkaloid biosynthesis, and demonstrate that nicotinic alkaloid content and particularly the nicotine content of plant cells that have the ability to produce nicotine it can be reduced by the sub-regulation of the expression A622.

Example 3: Construction of an inducible AR i A622 vector The constitutive suppression of A622 expression in the hairy roots of tobacco significantly inhibits root growth, by excluding the analysis of nicotinic alkaloids. To avoid this, a system of expression of the estradiol-inducible gene (XVE system) was developed. The XVE system produces RNAi hairpin molecules and deleted target genes only after the addition of an inducer (beta-estradiol) in the culture medium. The RNAi region containing antisense DNA fragments and sense A622 is excised from plasmid pHAN IB AL-X with Xho I and Xba I, and ligated into pBluescript KS which is digested with Xho I and Xba I. The RNAi region is then excised with Xho I and Spe I, and subcloned between the Xho I and Spe I sites in the MCS of the XVE vector pER8 (Zuo J. et al, Plant J., 24: 265-273 (2000)) to produce the vector binary pXVE-A622ARNÍ. The T-DNA region of pXVE-A622ARNi (See FIG. 7) contains a cassette for the inducible expression of estradiol of the chimeric transcription factor XVE, a selectable marker cassette hpt, and a cassette in which the expression of the A622 RNAi is low. control of the LexA-46 promoter, which is activated by XVE.

Example 4: Suppression of A622 in the hairy roots of tobacco The binary vector pXVE-A622AR i was introduced to the Agrobacterium rhizogenes strain 15834 by electroporation. The plants of N. tabacum cv. Petit Havana SRl were transformed by A. rhizogenes using a disk leaf method, as described by Kanegae et al, Plant Physiol. 105 (2) : 483-90. (1994). The hygromycin resistance (15 mg / L in medium B5) was used to select transformed roots. Transgenic hairy roots were grown at 27 ° C in the dark. The transgenic hair roots bearing the T-DNA of pXVE-A622ARNi were grown in medium B5 for 10 days and the silenced gene is induced by the addition of 17-beta-estradiol (2 μ?) For 4 days. RT-PCR analysis showed that the A622 expression is efficiently suppressed in the hairy roots of tobacco A8 and the A10 was transformed with the estradiol-inducible A622-suppressing constructs after the addition of estradiol. See figure 8A. The total RA was extracted from the hairy roots by using an RNeasy Plant mini kit (Qiagen). The cDNA was synthesized by using random hexamers and SuperScript strand priming synthesis system RT-PCR (Invitrogen). RT-PCR was carried out with 2.5 ng of the cDNA as a model using TaKaRa ExTaq (Takara Bio) under the following conditions: for the detection of A622, 22 cycles of 94 ° C for 30 seconds, 57 ° C for 30 seconds, and 72 ° C for 30 seconds; for the detection of -tubulin, 24 cycles of 94 ° C for 1 min, 52 ° C for 30 sec, and 72 ° C for 1 min.

Primers for the detection of A622; A622-077 5 'ATGGTTGTATC AGAGAAAAG A622-05R 5' CCTTCTGCCTCTATCATCCTCCTG Primers for the detection of α-tubulin; Tub RT-1 5 'AGTTGGAGGAGGTGATGATG Tub RT-2 5' TATGTGGGTCGCTCAATGTC The contents of nicotine were measured in hairy root lines transformed with an inducible A622 suppression construct without (-) and after induction of suppression with estradiol ( +). The RT-PCR plot in Figure 8B shows that the A622 expression is partially suppressed before the induction of estradiol. This is especially true with line # 8. The nicotine content varies between the suppressed A622 lines but became lower in the A622 suppressed lines than in the wild type hair roots.

Example 5: Identification of NBBl as a gene regulated by NIC sites A micro-configuration of cDNA prepared from a collection of cDNAs derived from Nicotiana sylvestris (atoh et al, Proc. Japan Acad., Vol. 79, Ser. B5 No. 6, pp. 151-154 (2003)) was used to search for novel genes which are controlled by nicotine biosynthesis regulated by NIC loci. The N. sylvestris cDNAs were amplified by PCR and stained on a mirror-coated slide (type 7 star, Amersham) by using a stain of Amersham Lucidea configuration. The DNA was immobilized on the surface of the slide by UV crosslinking (120 mJ / m2). Burley 21 N. tabacum (WT and nel nic2) seedlings were grown in medium B5 with half the strength supplemented with 1.5% (w / v) sucrose and 0.35% (w / v) gellan gum (Wako) in Agripot containers (Kirin). The roots of eight-week-old seedlings were harvested, immediately frozen with liquid nitrogen, and kept at -80 ° C until use. Total AR was isolated using a Plant RNeasy Mini kit (Qiagen) from the frozen roots, and the mRNA was purified using a GenElute Miniprep mRNA kit (Sigma). The cDNA was synthesized from 0.4 g of the purified mRNA by using a LabelStar Array kit (Qiagen) in the presence of Cy 3 or Cy 5 -dCTP. (Amersham). Hybridization of the cDNA in the microconfiguration slides and post-hybridization washes was carried out using a Lucida Pro hybrid machine (Amersham). The microconfigurations were scanned using a FLA-8000 scanner (Fujifilm). The acquired configuration images were quantified by the intensity of the signal with the ArrayGauge software (Fujifilm). The niclnic2 and wild type tobacco cDNA probes were labeled with Cy3 and Cy5 in reciprocal pair combinations. The hybridization signals were normalized by counting for the total signal strength of the pigments. The cDNA clones which hybridized to the wild-type probes more than twice as strongly compared to the nclnic2 probes were identified and these include NBBl. The full-length NBB1 cDNA was obtained by 5'- and 3'-RACE of the total N. tabacum RNA by using a SMART RACE cDNA amplification kit (Clontech). The nucleotide sequence of the NBB1 cDNA insert was determined in both strands using an ABI PRIS ® 3100 genetic analyzer (Applied Biosystems) and a BigDye® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). The nucleotide sequence of NBBl is set forth in SEQ ID NO: 3. The amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 4. The protein sequence includes a portion of the FAD linkage. A putative vacuolar signal peptide is located at the N terminal.

Example 6: Characterization of NBBl The NBB1 expression was investigated in tobacco plants by the analysis of the Northern blot technique. The RNA was extracted from the bodies of the plants which is treated with methyljasmonate vapor, using Nicotiana tabacum cv. Burley 21 (abbreviated below as T) and the niel, nic2 and nic2 niel mutants have mutations introduced in the Burley 21 background. The culture was in a sterile sealed environment, and the plants were elevated during 2 months at 25 ° C with 150 μ mole photons / m2 of light (16h light, 8h dark) in a medium 1A x B5 (3% sucrose, 0.3% gellan gum). The methyljasmonate treatment was completed by adding 0.5 mL of 100 μ? methyljasmonate to an Agripot container (Kirin, Tokyo) with a solid medium with a capacity of 80 cm3 and a gas capacity of 250 cm3 containing the plants. The treatment time was established at 0 hours and 24 hours. The parts of the root and the parts of the leaf (2a to 6a leaves of the body of the plant with a total of 7 to 10 leaves) were collected from the body of the plant and immediately frozen stored using liquid nitrogen. The RNA was extracted using an RNeasy Midi kit (Qiagen) according to the manufacturer's protocol. However, polyvinyl pyrrolidine was added at a concentration of 1% for the RLT. The operation of the column was carried out twice to increase the purity of the RNA. RNA staining was carried out according to the ordinary methods given by Sambrook and Russell (Sambrook, J. et ah, Molecular Cloning, Cold Spring Harbor Laboratory, Chapter 7 (2001)). The fragment of the 1278 bp sequence through the end (1759 bp) of the nucleotide sequence NBB1 (SEQ ID NO: 3) was used as the template of the probe. The template was prepared by amplifying the cDNA clone using PCR using the following primers: Primer 1: GGAAAACTAACAACGGAATCTCT Primer 2: GATCAAGCTATTGCTTTCCCT The probe was labeled with 32P using a Bcabest labeling kit (Takara) in accordance with the manufacturer's instructions. Hybridization was completed using ULTRAhyb (Ambion) as the buffer solution according to the manufacturer's protocol. The PMT probe was prepared from a PMT sequence cloned into a pADNcII vector in E. coli (Hibi et ah, 1994). The plasmid was extracted and purified from E. coli using a QIAprep Spin Miniprep kit (Qiagen), treated with the restriction enzymes Xbal and HindIII by the ordinary methods and run through agarose gel electrophoresis and around 1.5 kb of the DNA fragments were collected. A QIAquick gel extraction kit (Qiagen) was used for the collection. The collected DNA fragments were labeled with 32 P by the same methods used by the NBB1 probe and hybridized. The results are shown in Figure 9. As clearly shown in Figure 9, NBBl and PMT have the same pattern of expression in tobacco plants. The evidence that NBBl is involved in nicotine biosynthesis is that similar to PMT and A622, NBBl is under the control of NIC genes and these exhibit a similar pattern of expression for PMT and A622.

Example 7: Phylogenetic Analysis of NBBl The NBBl polypeptide has 25% identity and 60% homology to the bridged enzyme of berberine Eschscholzia californica (BBE). (Dittrich H. et ah, Proc.Nat.Accid Sel USA (Vol.88, 9969-9973 (1991)) An alignment of the NBBl polypeptide with EcBBE is shown in Figure 10. A phylogenetic tree is constructed using the sequence of the NBBl polypeptide and the BBE plant-like polypeptides (based on Carter and Thornburg, Plant Physiol., 134, 460-469 (2004).) The phylogenetic analysis was carried out using a close binding method with the CLUSTAL W. program. numbers indicate the values for own effort of the 1,000 replicates The sequences used were: EcBBE, opium poppy BBE of California (GenBank Accession No. AF005655), PsBBE, opium poppy (Papaver somniferum) probable reticulin oxidase (AF025430); BsBBE , Agracejina (Berberis stolonifera) BBE (AF049347); VuCPRD2, cowpea seed (Vigna unguiculata), drought-induced protein (AB056448), NspNEC5, Nicotiana sp., Nectarin V (AF503441 / AF503442), HaChox, sunflower (Helianthus annum), oxidase carbohydrate (AF47260 9); LsCHOX, lettuce (Lactuca sativa) carbohydrate oxidase (AF472608); and 27 arabidopsis genes (Atlg01980, Atlgll770, Atlg26380, Atlg26390, Atlg26400, Atlg26410, Atlg26420, Atlg30700, Atlg30710, Atlg30720, Atlg30730, Atlg30740, Atlg30760, Atlg34575, At2g34790, At2g34810, At4g20800, At4g20820, At4g20830, At4g20840, At4g20860, At5g44360, At5g44380, At5g44390, At5g44400, At5g44410, and At5g44440). The results are shown in Figure 11. The three known BBEs form a monophyletic group of separate organisms and are underlined and indicated as "real BBEs". The NBB1 sequence is not highly similar to any of the BBE or BBE type proteins, and they are separated from the other sequences at the base of the tree. Only the other type BBE protein of the genus Nicotiana, nectarine V, a protein described in the nectar of an ornamental hybrid Nicotiana langsdorffii X N. sanderae, Cárter and Thornburg (2004), branches with the protein induced by cowpea drought and various proteins of type Assumed BBE from Arabidopsis. Because the nectar of the alkaloids lacks ornamental tobacco and nectarine V has glucose oxidase activity, it is concluded that nectarine V is involved in antimicrobial defense in flowers and probably has no role in the synthesis of alkaloids. Id.

Example 8: Preparation of the NBBl deletion construct The 342-pb DNA fragment of the NBB1 cDNA is amplified by PCR and cloned into the vector pGE -T using the following primers.

Antisense Chain NBB1-20F-ECORI 5 'CCGGAATTCGCACAGTGGAATGAAGAGGACG 3' NBBl-18R-XhoI 5 'CCGCTCGAGGCGTTGAACCAAGCATAGGAGG 3' Sense chain NBB1-16F-CM 5 'CCATCGATGCACAGTGGAATGAAGAGGACG 3' NBB1-19R-Xbal 5 'GCTCTAGAGCGTTGAACCAAGCATAGGAGG 3' The resulting PCR products are run with EcoRI and Xhol by the antisense insert and with Clal and Xbal by insertion of the sense strand. The sense DNA fragment was subcloned into the pHA lBAL-X, followed by the insertion of the antisense fragment. The resulting plasmid contains an inverted repeat of the NBBl fragment, separated by the intron Pdk. The AR i region is excised from pHA NIBAL-X with BamH I and Sac I, and ligated into pBI121 to replace the GUS-encoded region and to produce the binary vector pARNi-NBBl. The T-DNA region of pHA IB AL-NBBl 3 '(See Figure 12) contains a cassette of the nptil selective marker and cassette for the expression of a hairpin RNAi with a double strand region corresponding to a 3' fragment of NBBl.

Example 9: Suppression of NBBl in tobacco BY-2 cells Treatment of methyl asmonate in tobacco BY-2 cells induces NBBl expression in the addition for gene expression by known enzymes in the path of nicotine biosynthesis . The effects of NBBl deletion are tested in BY-2 cells. The pARNi-NS5i vector was introduced into a strain A. tu efaciens EHA 105, which was used to transform tobacco BY-2 cells. The BY-2 cells were cultured in 100 ml of modified LS medium. Agrobacterium tumefaciens cells (100 μ?) In the YEB medium were added to 4 ml of BY-2 cells and cultured for 40 hours in the dark at 27 ° C. After infecting the tobacco cells they were washed with the modified LS medium, the washed tobacco cells are spread on the modified LS agar medium containing kanamycin (50 mg / 1) and carbenicillin (250 mg / 1). After 2 weeks in the dark at 27 ° C, the grown tobacco callus were transferred to a fresh LS selection medium with the same antibodies and cultured in the dark at 27 ° C for one more week. The grown tobacco cells were transferred to a liquid modified LS medium without the antibiotics. Transformed tobacco cells were subcultured at 7 day intervals. The cultured tobacco cells cultured with a modified LS medium are 2,4-D, at 27 ° C for 12 hours. After 100 μ? of methyljasmonate (MeJA) dissolved in DMSO was added to 100 mL of the tobacco suspension culture to give a final concentration of 50 μ ?, tobacco cells were cultured for an additional 48 hours. The cells treated with MeJA were filtered, collected and dried by freezing. The sulfuric acid, 3 ml of 0.1 N, was added to 100 mg of the sample dried by freezing. The mixture was sonic for 15 minutes, and filtered. A 28% ammonium solution was added to 1 ml of the filtrate and centrifuged for 10 minutes at 15,000 rpm. 1 ml of the supernatant was added to an Extrelut-1 column (Merck) and eluted with 6 ml of chloroform. The eluate was then added under reduced pressure at 37 ° C with an evaporator (Taitec TC-8 concentrator). The dry sample was dissolved in 50 μ? of an ethanol solution containing 0.1% dodecane. Gas chromatography (GC-14B) equipped with a capillary column and an FID detector was used to analyze the samples. A RESTEC Rtx-5Amina column (Restec) was used as the capillary column. The temperature of the column was maintained at 100 ° C for 10 min, elevated at 150 ° C at 25 ° C / min, maintained at 150 ° C for 1 min, raised to 170 ° C at ° C / min, maintained at 170 ° C for 2 min, raised to 300 ° C at 30 ° C / min, and then maintained at 300 ° C for 10 min. The injection and temperature of the detector was 300 ° C. A μ? of each sample was injected and the nicotinic alkaloids were quantified by the internal standard method. The accumulation of the nicotinic alkaloids followed by the elicitation of methyljasmonate was greatly reduced in the BY-2 cell lines suppressed by NBBl (N37 and N40) compared to the wild-type tobacco cells (See Figure 5). To determine whether the reduction of alkaloid accumulation in the NBB1-AR i lines is specifically related to the reduction of NBBL expression, preferably an indirect effect on the levels of gene expression for known enzymes in the path of Nicotine synthesis, levels of NBB1 expression and other genes were measured in treated lines of methyljasmonate, transgenic and control lines. The total RNA was isolated from the transgenic and wild type BY-2 cell lines which were treated with 50 uM MeJA for 48 h. The RNA levels of specific genes were determined by RT-PCR. The results are shown in Figure 6. In the silenced NBB1 lines the induction of NBB1 was not observed, but the induction of known genes from the nicotine biosynthetic pathway still occurs, as well as the induction of A622. Note also that the induction of NBBl does not affect the suppressed lines A662.

These results demonstrate that NBBl reductase is included in the path of nicotinic alkaloid biosynthesis, and that nicotinic alkaloid content and particularly, the nicotine content of plant cells that have the ability to produce nicotine can be decreased by the sub-regulation of NBBl expression.

Example 10: NBB1 suppression in the hairy tobacco roots The hairy SR-1 roots of tobacco accumulate nicotine as the major alkaloid. The effect of NBBl suppression on the alkaloid accumulation in its hair roots is studied. The binary vector pAR i-iVB .57 3 'was introduced into strain A. rhizogenes 15834 by electroporation. The plants N. tabacum cv. Petit Havana SRl were transformed by A. rhizogenes using a disk sheet method, as described above by the deletion of A622. in the hairy roots of tobacco. The hairy roots were selected and cultured and the alkaloids were extracted, purified and analyzed as described above. When the NBBl expression was suppressed by RNAi, the roots of the transgenic roots (HN6, HN19, HN20 and HN29) contain highly reduced levels of nicotine compared to the control cell line, as well as the reduced levels of anatabine (see figure 13). ). The transgenic hair roots carrying pHANNIB AL-NBBl 3 'were grown in the B5 medium for two weeks and the expression of the gene was analyzed by RT-PCR. The expression NBBl was specifically suppressed in four transgenic lines (See Figure 14). The NBB1 expression of other genes for the enzymes in the nicotine biosynthetic pathway was not affected. The results show that the results of reduced nicotine accumulation of reduced NBBl expression do not lack the expression of genes for known enzymes of the nicotine biosthesis pathway.

Example 11: NBBl deletion in transgenic tobacco plants Two fragments attB-NBBl were amplified by PCR of the NBBl cDNA in the pGEM-T vector using a set of primers of NBBl-aatBl and adapted attBl and a conjugate NBB1-attB2 and adapted attB2. Gene-specific primers: NBBl-attBl 5 'AAAAAGCAGGCTTCGAAGGAGATAGAACCATGGTTCCGCTCATAATTCTGATCAGC TT NBBl-attB2 51 AGAAAGCTGGGTCTTCACTGCTATACTTGTGCTCTTGA Adapted primers: attBl adapted 5 'GGGGAC AAGTTTGTACAAAAAAGCAGGCT adapted attB2 5' GGGGACC ACTTTGTACAAGAAAGCTGGGT The PCR conditions used were those recommended by the manufacturer. An entry of clone pDONR221-NBBl-l was created by the recombination reactions BP between the products attB-NBBl PCR and pDONR221 (Invitrogen). The NBB1 ORF was transferred from vector pDONR221-NBB1-l to a binary vector GATEWAY pANDA 35HK which was designated to express a dsAR with a partial GUS fragment under the CaMV35S promoter (Dr. Ko Shimamoto, NAIST) by the LR reaction. The resulting NBB1 vector of RNAi is referred to as a complete pANDA-NBB1. The complete PANDA-NBB 1 T-DNA (See Figure 15) contains a nptll selectable marker cassette, a NBB1 cassette of RNAi in which the full-length encoded region of NBB1 is presented in the inverted repeats separated by GUS-linked, and a selectable labeled cassette hpt. The complete binary pANDA-NBB1 vector was introduced to a strain A. tumefaciens EHA105 by electroporation. The plants N. tabacu cv. Petit Havana SRl were transformed by A. tumefaciens using a leaf-disk method, basically as described by Kanegae et al, Plant Physiol. 105, 483-490 (1994). The hygromycin resistance (30 mg / L in the MS medium) was used for the selection. The transgenic plants were regenerated from the disk leaves as described and grown at 27 ° C under light conditions in a growth chamber. The tissue of the leaf was collected from the growth of the plants of the T0 generation during 36 days. The alkaloids were extracted, purified and analyzed as described above. The levels of nicotine in the leaves of the plants of the lines transformed with the vector of suppression NBBl pA DA-NBBl complete were reduced compared with the wild type (See figure 16). The leaves of the transgenic lines (# 6), # 14 and # 22) contain nicotine levels only about 16% of the level in the leaves of wild type plants. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (70)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An isolated nucleic acid molecule, characterized in that it comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence set out in SEQ ID NO: 3; (b) a nucleotide sequence comprising a sequence of more than 90% identical to at least 15 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 3; (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence set forth in SEQ ID NO: 4; and (d) a nucleotide sequence encoding a polypeptide having an amino acid sequence with at least 80% similarity for SEQ ID NO: 4 and NBB1 activity.
2. 2. A nucleic acid construct, characterized in that it comprises, in the 5 'to 3' direction, a promoter operably linked to a heterologous nucleic acid encoding at least a portion of the NBB1 in sense or antisense orientation, and a terminator.
3. 3. A nucleic acid construct, characterized in that it comprises a short interfering RNA that suppresses NBBl expression.
4. 4. A nucleic acid, characterized in that it comprises a sequence of chimeric RNA / DNA oligonucleotides with at least 90% similarity for at least 15 consecutive nucleotides of SEQ ID NO: 3.
5. 5. A plant cell, characterized in that it comprises the nucleic acid construct according to claims 1-3.
6. 6. A method for producing a tobacco plant reduced in alkaloid, characterized in that it comprises genetically engineering the suppression of NBBl in the plant.
7. The method according to claim 6, characterized in that the engineering comprises introducing into the plant cell a nucleic acid construct comprising a short interfering RNA that suppresses the expression of NBBl.
8. The method according to claim 6, characterized in that the engineering comprises introducing into the cell of the plant plant an enzyme RNA molecule that unfolds a transcript of NBBl mRNA.
9. 9. The method according to claim 6, characterized in that engineering comprises introducing into the cell of the plant plant a nucleic acid construct, comprising, in the 5 'to 3' direction, a promoter operably linked to a heterologous nucleic acid encoding at least a portion of the NBB1 in sense or antisense orientation, and a terminator.
10. The method according to claim 6, characterized in that the engineering comprises inducing a directed mutation in an endogenous NBB1 sequence through the introduction of chimeric RNA / DNA oligonucleotides.
11. 11. A method for reducing an alkaloid in a tobacco plant, characterized in that it comprises (a) suppressing NBBl; and (b) suppressing at least one additional nicotine biosynthesis enzyme selected from the group consisting of aspartate oxidase, quinolinate synthase, quinolate phosphoribosyl transferase, orinithine decarboxylase, putrescine N-methyltransferase, and methylpi threecin oxidase, and A622.
12. 12. The method according to claim 11, characterized in that NBB1 and putrescine N-methyltransferase are suppressed.
13. The method according to claim 11, characterized in that NBBl, A622, and putrescine N-methyltransferase are deleted.
14. The method according to claim 11, characterized in that NBB1 and phosphoribosyltransferase quinolate are deleted.
15. 15. The method according to claim 11, characterized in that NBB1, A622, and quinolate phosphoribosyl transferase are suppressed.
16. 16. A tobacco plant characterized by having a suppression of NBBl produced by genetic engineering and a reduced total alkaloid content.
17. 17. The tobacco plant reduced in alkaloid according to claim 16, characterized because nicotine is reduced.
18. 18. The reduced tobacco plant in alkaloid according to claim 16, characterized in that the anatabine is reduced.
19. 19. The tobacco plant reduced in alkaloid according to claim 16, characterized in that the anabasine is reduced.
20. 20. A genetically engineered tobacco plant, characterized in that it comprises a chimeric nucleic acid construct comprising the nucleic acid molecule according to claim 35 linked to a heterologous nucleic acid.
21. 21. The genetically engineered tobacco plant according to claim 20, characterized in that the nucleic acid molecule according to claim 1 is linked to a heterologous DNA in an antisense orientation.
22. 22. The genetically engineered tobacco plant according to claim 41, characterized in that the nucleic acid molecule according to claim 1 is linked to a heterologous DNA in a sense orientation.
23. 23. Progeny of the plant according to claims 18-19, characterized in that the progeny have suppression of NBBl.
24. 24. A tobacco plant produced by genetic engineering by the method according to claim 11, characterized in that the plant is characterized by a reduction in the content of an alkaloid, suppression of NBBl, and suppression of at least one biosynthesis enzyme of additional nicotine selected from the group consisting of aspartate oxidase, quinolinate synthase, quinolate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyltransferase, and methylputrescin oxidase, and A622.
25. 25. The tobacco plant produced by genetic engineering according to claim 24, characterized in that the plant is characterized by a total reduction in the alkaloid content.
26. 26. The tobacco plant produced by genetic engineering according to claim 24, characterized in that the plant is characterized by a reduction in nicotine.
27. 27. The tobacco plant produced by genetic engineering according to claim 24, characterized in that the plant is characterized by a reduction of anatabine.
28. 28. The tobacco plant produced by genetic engineering according to claim 24, characterized in that the plant is characterized by a reduction of anabasine.
29. 29. Seeds of a tobacco plant according to claims 24-28.
30. 30. A tobacco product, characterized in that it comprises a portion of a tobacco plant according to claims 24-28.
31. 31. A nicotine reduced tobacco product, characterized in that it comprises a portion of a tobacco plant according to claims 24-28.
32. 32. A tobacco product reduced in anatabine, characterized in that it comprises a portion of a tobacco plant according to claims 24-28.
33. 33. A tobacco product reduced in anabasine, characterized in that it comprises a portion of the tobacco plant according to claims 24-28.
34. 34. A product of tobacco reduced in alkaloids, characterized in that it has a reduced collective quantity of N '-nitrosonornicotine (NNN), 4-methylnitrosoamino-1- (3-pyridyl) -1-butanone (NNK),' -nitrosoanatabine (NAT) and '-no trosaanabasina (NAB) and which is prepared from tobacco from a plant genetically engineered for NBBl suppression, the reduced collective amount of NNN, NNK, NAT and NAB is reduced in relation to the amount in a similar tobacco product prepared from a control tobacco plant.
35. 35. The tobacco product according to claims 30-34, characterized in that the tobacco product is a cigarette.
36. 36. A smoking cessation product, characterized in that it comprises a portion of the tobacco plant according to claims 24-28.
37. 37. An isolated NBBl polypeptide, characterized in that it has an amino acid sequence set forth in SEQ ID NO: 4.
38. 38. A polypeptide, characterized in that it has a sequence which is a variant of SEQ ID NO: 4.
39. 39. An NBBl enzyme isolated encoded by a nucleic acid sequence, characterized in that it is selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID NO: 3; (b) a nucleotide sequence comprising a sequence of more than 90% identical to at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 3 encoding an NBBl enzyme; (c) nucleic acid sequences that hybridize to SEQ ID NO: 3 under conditions of moderate severity or high severity and encode an NBB1 enzyme; and (d) nucleic acid sequences that differ from the nucleic acid sequence of (a), (b) or (c) above due to the degradation of the genetic code and encodes an NBBl enzyme.
40. 40. A nucleic acid construct, characterized in that it comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence comprising a sequence of more than 90% identical to at least 15 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1; (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; and wherein the nucleotide sequence is operably linked in sense or antisense orientation to an active heterologous promoter in the plant cell.
41. 41. A plant cell, characterized in that it contains the nucleic acid construct according to claim 40.
42. 42. A method for producing a tobacco plant reduced in alkaloids, characterized in that it comprises genetically engineering the suppression of A622 in the plant .
43. 43. The method according to claim 42, characterized in that engineering comprises introducing into the plant cell a nucleic acid construct comprising a short interfering RNA that suppresses the expression of A622.
44. 44. The method according to claim 42, characterized in that the engineering comprises introducing into the cell of the plant plant an enzyme RNA molecule that unfolds the A622 mRNA transcript.
45. 45. The method according to claim 42, characterized in that the engineering comprises introducing into the cell of the plant plant a nucleic acid construct, comprising, in the 5 'to 3' direction, a promoter operably linked to a heterologous nucleic acid encoding at least a portion of A622 in sense or antisense orientation, and a terminator.
46. 46. The method according to claim 42, characterized in that engineering comprises inducing a directed mutation in an endogenous A622 sequence through the introduction of chimeric RNA / DNA oligonucleotides.
47. 47. The method according to claim 42, characterized in that the alkaloid is nicotine.
48. 48. The method according to claim 42, characterized in that the alkaloid is anatabine.
49. 49. The method according to claim 42, characterized in that the alkaloid is anabasine.
50. 50. A method for reducing an alkaloid in a tobacco plant, characterized in that it comprises (a) suppressing A622; and (b) suppressing an additional nicotine biosynthesis enzyme selected from the group consisting of aspartate oxidase, quinolinate synthase, quinolate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyltransferase, and methylprorescin oxidase.
51. 51. The method according to claim 50, characterized in that A622 and putrescine N-methyltransferase are suppressed.
52. 52. The method according to claim 50, characterized in that it comprises suppressing A622, and phosphoribosyltransferase quinolate.
53. 53. A tobacco plant produced by genetic engineering, characterized in that it comprises a chimeric nucleic acid construct according to claim 58. 5.
54. The tobacco plant produced by genetic engineering according to claim 53, characterized in that the nucleotide sequence is in an antisense orientation.
55. 55. The tobacco plant produced by genetic engineering according to claim 53, characterized in that the nucleotide sequence is in a sense orientation.
56. 56. Progeny of the plant according to claim 53, characterized in that the progeny have suppression of A622.
57. 57. A tobacco plant characterized by having a suppression of A622 and reduced alkaloid content.
58. 58. The reduced tobacco plant according to claim 57, characterized in that the nicotine is reduced.
59. 59. The tobacco plant according to claim 57, characterized in that the anatabine is reduced.
60. 60. The tobacco plant according to claim 57, characterized in that the anabasine is reduced.
61. 61. A tobacco plant produced by genetic engineering produced according to the method of claim 51, characterized in that the plant is characterized by suppressing A622 and putrescine timethyltransierase.
62. 62. The tobacco plant produced by genetic engineering produced by the method according to claim 51, characterized in that the plant is characterized by suppressing A622 and quinolate phosphoribosyl tansferase.
63. 63. Seeds of a plant in accordance with claims 52-62.
64. 64. A tobacco product, characterized in that it comprises the tobacco plant according to claims 53-62.
65. 65. A nicotine-reduced tobacco product, characterized in that it comprises the tobacco plant according to claims 53-62.
66. 66. A product of tobacco reduced in alkaloid, characterized because it has a reduced collective quantity of '-nitrosonornicotine (NNN), 4-methylnitrosoamino-1- (3-pyridyl) -1-butanone (NNK), N'-ni trosoanatabina ( NAT) and N '-ni-trosaanabasin (NAB) and which is prepared from tobacco of a plant produced by genetic engineering for NBBl suppression, the reduced collective amount of NNN, NNK, NAT and NAB is reduced in relation to the amount in a similar tobacco product prepared from a control tobacco plant.
67. 67. The tobacco product characterized in that it has a reduced amount of nitrosoanatabin (NAT) prepared from a tobacco plant having an A622 suppression produced by genetic engineering, the amount of NAT being reduced relative to the level in a similar tobacco product prepared of a non-transformed control tobacco plant.
68. 68. A tobacco product reduced in anabasine prepared from a tobacco plant that has suppression of A622 produced by genetic engineering, characterized in that the amount of NAT is reduced relative to the level in a similar tobacco product prepared from a tobacco plant of control not transformed.
69. 69. The tobacco product according to claims xx-xx, characterized in that the tobacco product is a cigarette.
70. 70. A smoking cessation product, characterized in that it comprises a portion of the tobacco plant according to claims 53-62.