HK1233677A1 - A method for reducing alkaloids in a plant, a genetically engineered cell for reducing alkaloids in a plant and a reduced-alkaloids product - Google Patents

Description

Method for reducing alkaloids in plants, genetically engineered plant cells and reduced alkaloid products

The application is a divisional application of an invention application with the application number of 200680010544.X (International application number of PCT/IB2006/001741), the application date of 2006-02-28 and the invention name of "reduction of nicotine alkaloid level in plants".

Benefit of provisional application

Priority is granted to U.S. provisional application No. 60/656,636, filed on 28.2.2005, the contents of which are hereby incorporated by reference in their entirety.

Technical Field

The present invention relates to the field of molecular biology and down-regulation of alkaloid synthesis. In particular, the present invention relates to methods and constructs for reducing nicotine alkaloids (nicotinicallkalidoids) in plants, particularly but not exclusively tobacco plants.

Background

Currently, several methods exist for reducing nicotine alkaloids such as nicotine in plants. For example, low nicotine tobacco lines have been used as breeding stocks of low nicotine cultivars. Legg et al, Crop Sci 10:212 (1970). Genetically engineered methods can also be used to reduce nicotine levels. For example, U.S. Pat. nos. 5,369,023 and 5,260,205 discuss reducing nicotine levels by antisense targets of endogenous Putrescine Methyltransferase (PMT) sequences. Voelckel et al, biotechnology 11: 121-. The tobacco quinolate (quinolate) phosphoribosyltransferase (QPT) gene has been cloned, Sinclair et al, Plant MoI.biol.44:603-617(2000), and its antisense inhibition provided a significant reduction in nicotine in transgenic tobacco plants. Xie et al, Tobacco Science progress (Recent Advance sin Tobacco Science)30:17-37 (2004). See also U.S. patent nos. 6,586,661 and 6,423,520.

Several nicotine biosynthetic enzymes are known. See, for example, Hashimoto et al, Plant MoI.biol.37:25-37 (1998); reichers and Timko, Plant MoI.biol.41:387-401 (1999); imanishi et al, plantaMoI.biol.38: 1101-1111 (1998). There is a continuing need for additional genetic engineering methods to reduce nicotine alkaloids even further. For example, when only PMT is down-regulated in tobacco, nicotine is reduced but anatabine is increased by approximately 2-6 times. Chintapaker and Hamill, Plant MoI. biol 53:87-105 (2003); steppuhn et al, PLoS Biol 2(8), e217:1074-1080 (2004). When only QPT is down-regulated, there are still significant amounts of alkaloids. See U.S. plant variety certificate No. 200100039.

Reducing the content of total alkaloids in tobacco will increase the value of tobacco as biomass resource. Tobacco produces 8 tons more dry weight per acre when grown under conditions of maximum biomass such as high density and multiple harvests (multiple crops) compared to other crops used as biomass. However, large-scale planting and processing of conventional tobacco biomass has several disadvantages. For example, since conventional tobacco biomass contains about 1% to about 5% alkaloids depending on the species, significant time and energy is spent extracting, separating, and processing the tobacco alkaloids. Conventional tobacco biomass contains up to about 800 pounds of alkaloids per acre. Meanwhile, people handling tobacco may suffer from excessive exposure to nicotine, commonly referred to as "green tobacco disease".

Reduced alkaloid tobacco is more amenable to non-conventional purposes such as biomass and derived products (derivdroducts). For example, the use of reduced alkaloid tobacco for ethanol production and protein by-products (co-products) is advantageous. U.S. published application No. 2002/0197688. In addition, the alkaloid-free tobacco or components thereof can be used as a forage crop, animal feed, or human nutritional source. And Id.

In addition to these benefits, the concomitant reduction in nicotine requires a more successful approach to assist smokers in quitting smoking. Nicotine Replacement Therapy (NRT) is not very effective as a smoking cessation therapy because its success rate is less than 20% 6 to 12 months after 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 (addition) 90:31-42 (1995). Reduced or no nicotine cigarettes have helped smokers successfully quit smoking, giving them nicotine but allowing them to have a habit of smoking. In addition, the cigarette without nicotine can relieve smoking addiction and other smoking abstinence symptoms. See Rose, Psychopharmacology (Psychopharmacology)184:274-285(2006) and Rose et al NicotineTobacco Res.8:89-101 (2006).

Thus, there is a continuing need to identify additional genes that can act on the expression of such genes to reduce nicotine alkaloid levels.

Disclosure of Invention

Can act on two genes of A622 and NBBl to realize the reduction of the nicotine alkaloid level in the plants. In particular, inhibition of one or both of a622 and NBBl can be used to reduce nicotine in tobacco plants.

Thus, in one aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of (a) the nucleotide sequence set forth in SEQ ID NO: 3; (b) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO. 4; and (c) a nucleotide sequence that differs from the nucleotide sequence of (a) or (b) due to the degeneracy of the genetic code and encodes a polypeptide having NBBl expression.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of (a) the nucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO. 2; and (c) a nucleotide sequence that differs from the nucleotide sequence of (a) or (b) due to the degeneracy of the genetic code and encodes a polypeptide having A622 expression, wherein the nucleotide sequence is operably linked to a heterologous promoter.

In another aspect, the invention provides a method of reducing an alkaloid in a plant, the method comprising reducing NBBl and a622 expression.

In another aspect, the invention provides a transgenic plant having reduced a622 expression and alkaloid content, and also provides a tobacco plant having reduced NBBl expression and alkaloid content. The invention also provides a genetically engineered plant with reduced nicotine and anatabine content.

In another aspect, the invention provides a reduced nicotine smoking article made from a tobacco plant having reduced expression of a622 or NBBl.

Drawings

Figure 1 depicts a nicotine biosynthetic pathway. Abbreviated as AO aspartate oxidase, QS quinolinate synthase, QPT quinolinate phosphoribosyltransferase, ODC ornithine decarboxylase, PMT putrescine N-methyltransferase, and DAO diamine oxidase;

FIG. 2A schematically illustrates pHANNIBAL;

FIG. 2B schematically illustrates pHANNIBAL-X in which the multiple linker (polylinker) site has been modified, i.e., pHANNIBAL-X in which there is an improved multiple linker site;

FIG. 3 depicts a strategy for making plant RNAi binary vectors using modified pHANNIBAL-X as an intermediate plasmid;

FIG. 4 depicts the T-DNA region of pRNAi-A622;

FIG. 5 depicts the accumulation of nicotine alkaloids in BY-2 cells, A662 silenced BY-2 cells, and NBBl silenced BY-2 cells, wherein nicotine alkaloids in BY-2 cells; WT-wild type, non-transformed cells; vector-cells transformed with marker only; a3, a21, a33, a 43-cells transformed with a construct for inhibiting a 622; n37, N40 cells transformed with a construct for inhibiting NBB 1;

FIG. 6 depicts the expression of genes for A622, NBBl and known enzymes of the nicotine biosynthetic pathway in wild type BY-2 cells, A622 silenced BY-2 cells and NBBl silenced BY-2 cells, wherein the expression of genes for A622, NBB1 and other known enzymes of the nicotine biosynthetic pathway in the A622 and NBB1 silenced BY-2 lines; a3, a21, a33 and a43 are a622 silenced lines; n37 and N40 are NBB1 silent lines; WT was non-transformed control; n is a negative control;

FIG. 7 depicts the T-DNA region of inducible A622 expression vector pXVE-A622 RNAi;

FIG. 8A depicts the specific inhibition of A622 in hairy root lines transformed with an inducible A622 inhibition construct following estradiol-induced inhibition;

FIG. 8B illustrates reduced nicotine levels in hairy root lines transformed with the inducible A622 suppression construct after estradiol-induced suppression; its expression of a622PMT and tubulin as detected by RT-PCR (fig. 8A) and nicotine content in tobacco hairy root lines (fig. 8B); the tobacco hairy root line is cultured for 4 days on a liquid culture medium containing (+) or not containing (-) additional estradiol, and then the root is harvested and analyzed; WT-non-transformed wild type lines; XVE-A622RNAi #8 and XVE-A622RNAi #10 are inducible RNAi strains.

FIG. 9 depicts northern blot analysis of NBBl expression and PMT expression in root and leaf tissues of wild type tobacco as well as of nicl, nic2 and niclnic2 mutants;

FIG. 10 depicts an alignment of NBB1 with the Berberine bridging enzyme (bridgeenzyme) (EcBBE) of Linnaeus (Eschscholzia californica);

FIG. 11 depicts a phylogenetic tree constructed using NBB1 and plant BBE-like (BBE-like) protein sequences;

FIG. 12 depicts the T-DNA region of NBBl inhibitory vector pHANNIBAL-NBBl 3';

figure 13 depicts the reduction of nicotine alkaloid synthesis in NBBl inhibited tobacco hairy roots, wherein nicotine alkaloid production from tobacco hairy root lines. Wild type-hairy root strain transformed with wild type agrobacterium rhizogenes; vector 1 and vector 2-hairy root lines generated by transformation with vectors without NBB1 sequence; HN6, HN19, HN20, HN 29-hairy root lines transformed with NBB1 inhibition vector pRNAi-NBB 13';

figure 14 depicts expression of known enzymes involved in NBB1, a622 and nicotine biosynthesis in NBBl silenced hairy root lines and control hairy root lines: n6, N19, N20, N29 vector control VC;

FIG. 15 depicts the full-length T-DNA region of NBBl suppression vector pANDA-NBB 1;

FIG. 16 depicts nicotine levels in leaves of tobacco (Nicotiana tabacum) plants from lines transformed with NBBl suppression vector pANDA-NBBl full length.

Detailed Description

The inventors of the present invention have identified two genes, a622 and NBBl, which can be used to effect a reduction in nicotine alkaloid levels in plants including, but not limited to, tobacco. Although A622 has been previously identified by Hibi et al plant cells (plantaCell) 6:723-735(1994), the present inventors have discovered a previously unknown effect of A622, herein to reduce nicotine biosynthesis. Prior to the present inventors' discovery, NBBl was not known at all in the art; according to the present invention, the inventors of the present invention also elucidated the role of NBBl in a pathway for reducing nicotine alkaloid content in plants.

Accordingly, the invention includes methods and constructs for reducing nicotine alkaloid levels in plants by inhibiting expression of a622 or NBBl. That is, nicotine alkaloid levels can be reduced by inhibiting one or both of a622 and NBBl. According to this aspect of the invention, the plant or any part of the plant is transformed with a nucleotide sequence, the expression of which inhibits at least one of a622 and NBBl and reduces nicotine alkaloid content.

In another aspect of the invention, nicotine can be further inhibited in plants by simultaneously inhibiting the expression of any known enzyme of the nicotine biosynthetic pathway such as QPT or PMT and at least one of a622 and NBBl. For example, in addition to nicotine reduction, the present invention provides a means of reducing anatabine simultaneously. Thus, by inhibiting nicotine biosynthesis genes such as QPT and at least one of a622 and NBBl, anatabine levels can be reduced.

Consistent with the present invention, a number of alkaloid reduced plants and by-products may be obtained by methods that affect the expression of a622 and/or NBBl until the final reduction of nicotine alkaloid content in the plant. For example, tobacco plants that have inhibited expression of a622 or NBBl can be used to produce reduced nicotine cigarettes, which can be used as smoking cessation products. Likewise, reduced-nicotine tobacco can be used as a forage crop, animal feed, or human nutritional source.

Other objects, features and advantages of the present invention will be apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Definition of

Technical terms used in the present specification are commonly used in biochemistry, molecular biology and agriculture; therefore, they can be understood by those skilled in the art to which the present invention pertains. Those technical terms can be found, for example, in the following documents: molecular cloning: a guide to the experiment (MOLECULAR CLONING: A LABORATORY MANUAL), third edition, Vol.1-3, compiled by Sambrook and Russel, Cold Spring Harbor LABORATORY Press, Cold Spring Harbor (Cold Spring Harbor), N.Y., 2001; molecular biology existing design of experiments (Current PROTOCOLS IN moleculalarrylogy), authored by Ausubel et al, Greene Publishing Associates and Wiley-Interscience, New York,1988 (periodic updates); molecular biology short experimental design: summary OF the design METHODS OF the existing experiments IN MOLECULAR BIOLOGY (SHORT PROTOCOLS IN MOLECULAR BIOLOGY: A COMPENDIUM OF METHODS FROM CURRENT PROTOCOLS IN MOLECULAR BIOLOGY), 5 th edition, Vol.1-2, eds OF Ausubel et al, John Wiley & Sons, Inc., 2002; genome analysis: experimental guidelines (GENOME ANALYSIS: A LABORATORY MANUAL), Vol.1-2, Main eds of Green et al, Cold Spring Harbor LABORATORY Press, Cold Spring Harbor, N.Y., 1997.

Methods involving plant biology techniques are described herein and are described in detail as plant molecular biology methods: a method strategy section of the Experimental Process guide (METHODS IN PLANT MOLECULAR BIOLOGY: A LABORATORYCOURSE MANUAL) (eds. by Maliga et al, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1995). Various techniques using PCR, for example, the PCR experiment design method AND the application GUIDE (PCR PROTOCOLS A GUIDE TO METHODS AND APPLICATIONS) (Academic Press San Diego,1990) under the Innis et al, AND the PCR primers under the major editors of Dieffenbach AND Dveksler: experimental guidelines (PCR PRIMER: A LABORATORY MANUAL) (second edition, Cold Spring Harbor LABORATORY Press, Cold Spring Harbor, N.Y.,2003) are described. PCR Primer pairs can be derived from known sequences by known techniques, such as using computer programs for this purpose (e.g., Primer, Version 0.5,1991, Whitehead Institute for biological Research, Cambridge), methods MA. for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Caruthers, tetra.letters.22: 1859-1862(1981) and Matteucci and Caruthers, J.am.chem.Soc.103:3185 (1981).

Restriction enzyme digestion, phosphorylation, ligation and transformation were performed as described in the MOLECULAR CLONING protocols (MOLECULAR CLONING: A LABORATORY MANUAL) (Sambrook et al, second edition (1989), Cold spring harbor LABORATORY Press). Unless otherwise indicated, all reagents and materials used for bacterial cell growth and maintenance are available from Aldrich Chemicals (Milwaukee Wis.), DIFCO Laboratories (Detroit, Mich.), Invitrogen (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.).

A622 expression is controlled by the genes NIC1 and NIC2 located in tobacco plants, Hibi et al, plant cells (the plant Cell), 6:723-735 (1994). A622 expression has been reported to be identical to PMT. Shoji, T, et al, Plant Cell physiology (Plant Cell physiology), 41:9: 1072-; shoji T, et al, Plant MoI Biol,50:427-440 (2002). Both A622 and PMT are expressed specifically in roots, particularly in the cortex and endothelial layers of root and root hair tip portions (apicalparts), Shoji et al (2002). Furthermore, a622 and PMT are expressed in the same manner as NIC regulation and methyl-jasmonate stimulation. As a traumatic response to aerial tissue, a622 was induced in the roots of tobacco (Nicotiana tabacum). Cane et al, functional plant Biology (functional plant Biology), 32,305-320 (2005). Under conditions that lead to QPT induction, a622 was induced in wounded leaves of pink blue tobacco (n. 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 nucleic acid sequence encodes a protein (SEQ ID NO:2) that is similar to isoflavone reductase (IFR) and comprises an NADPH-binding motif (motif). A622 showed homology to TP7, TP7, a phenylcoumaran benzylic ether reductase (PCBER) involved in lignin biosynthesis. Shoji et al (2002), supra. However, when A622 was subjected to expression analysis in E.coli using two different substrates, no PCBER activity was observed.

Based on the co-regulation of a622 and PMT and the similarity of a622 to IFR, it is proposed that a622 functions as a reductase in the final step of nicotine alkaloid synthesis. Hibi et al (1994); shoji et al (2000 a). However, when the protein was expressed in bacteria (id.), no IFR activity was observed. The function of a622 was previously unknown, and a622 was not known to play a role in nicotine synthesis before that.

A622 expression refers to the biosynthesis of the gene product encoded by SEQ ID NO. 1. A622 inhibition refers to a decrease in a622 expression. A622 inhibition has the ability to down-regulate the nicotine alkaloid content in plants or plant cells.

Alkaloid is a nitrogen-containing basic compound found in plants and is produced by secondary metabolism. Nicotine alkaloid is nicotine or an alkaloid structurally related to nicotine and synthesized from compounds produced in the nicotine biosynthetic pathway. In the case of tobacco, nicotine alkaloid content and total alkaloid content are used synonymously.

Exemplary tobacco alkaloids include, but are not limited to, nicotine, demethylnicotine, anatabine, anabasine, anatabine (anadaline), N-methylacetazone, N-methylanabasine, Masmine nicotine, pseudoscouring (anabaseine), N '-formylnornicotine (N' -formarnicotine), diennicotin, and cotinine. Other very few alkaloids in tobacco are reported, for example, in Hecht, S.S. et al, chemical Research report (Accounts of chemical Research)12:92-98 (1979); tso, t.c., Production, Physiology and Biochemistry of Tobacco plants, Ideals inc., Beltsville, MD (1990). The chemical structures of several alkaloids are described, for example, in Felpin et al, J.org.chem.66:6305-6312 (2001).

Nicotine is the major alkaloid in tobacco (n.tabacum) and also in 50-60% of other species in the genus nicotiana. Demethylated nicotine, anatabine, and quinhydrozine are the other most important alkaloids in tobacco (n. Of any, anatabine is generally not the major alkaloid but does accumulate to relatively high amounts in 3 species; chenopodine is the major alkaloid in 4 species. Nornicotine is the major alkaloid in 30-40% of the nicotiana species. Depending on the variety, about 85% to about 95% of the total alkaloids in tobacco are nicotine. Bush, l.p., Tobacco products, Chemistry and Technology (tobaco Production, Chemistry and Technology), Coresta285-291 (1999); hoffmann et al Journal of Toxicology and environmental Health (Journal of Toxicology and environmental Health), 41:1-52, (1994).

In the present invention, nicotine alkaloid content in genetically engineered plants can be reduced by down-regulating at least one of a622 and NBBl. In addition, nicotine alkaloid content can be reduced by down-regulating one of the nicotine biosynthetic enzymes such as QPT or PMT and at least one of both a622 and NBBl.

Anatabine is a nicotine alkaloid. Previous studies have demonstrated that PMT inhibition reduces nicotine levels but increases putrescine and anatabine levels. Chintapaker and Hamill, Plant MoI. biol.53:87-105 (2003); sato et al, Proc.Natl.Acad.Sci.USA 98, 367-; steppuhn, A. et al, PLoSBOL 2(8): e217:1074-1080 (2004). For the purposes of the present invention, the anatabine content of genetically engineered plants can be reduced by down-regulating at least one of A622 and NBBl. Anatabine levels can be further reduced by down-regulating one of the nicotine biosynthetic enzymes such as QPT and at least one of both a622 and NBBl.

BY-2 tobacco cells are a cell line established in the 60 s of the twentieth century BY Nicotiana japonica Co., Ltd, and are derived from the tobacco variety Brilliant Yellow-2 (Bright Yellow-2). Since this cell line grows rapidly during tissue culture, it is readily propagated in large quantities and used for genetic manipulation. BY-2 tobacco cells are widely used as a model plant cell line for basic research. BY-2 tobacco cells do not produce nicotinic alkaloids when cultured in standard media. The addition of jasmonate to the medium induces the production of nicotinic alkaloids.

Complementary DNA (cDNA) is a single-stranded DNA molecule formed by enzymatic reverse transcription using mRNA as a template. One skilled in the art also uses "cDNA" to refer to a double-stranded DNA molecule that includes the single-stranded DNA molecule and its complementary DNA strand. Primers that are generally partially complementary to the mRNA are used to initiate the reverse transcription process to produce cDNA.

Expression refers to the biosynthesis of a gene product. With respect to structural genes, for example, expression involves transcription of the structural gene into mRNA and translation of the mRNA into one or more polypeptides.

A gene refers to a polynucleotide sequence that includes the regulatory and coding sequences required for the production of a polypeptide or precursor. The polypeptide can be encoded by the full length coding sequence or any portion of the coding sequence. The gene may consist of an uninterrupted coding sequence or it may also comprise one or more introns, joined by appropriate splice junctions. Furthermore, a gene may comprise one or more modifications in the coding or untranslated regions that may affect the biological activity or chemical structure of the expression product, the rate of expression, or the manner in which expression is regulated. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides. In this regard, the modifier gene may be referred to as a "variant" of the "native" gene.

Genetic Engineering (GE) includes any method of introducing nucleic acids or specific mutations into a host organism. For example, when a tobacco plant is transformed with a polynucleotide sequence that inhibits the expression of a gene such as a622 or NBBl, and thus reduces nicotine levels, the tobacco plant is genetically engineered. In contrast, a tobacco plant that is not transformed with a polynucleotide sequence that inhibits expression of a target gene is a control plant and is referred to as a "non-transformed" plant.

In this context, the term "genetic engineering" encompasses "transgenic" plants and plant cells (see definitions, below) as well as plants and plant cells produced by targeted mutagenesis, for example, by using chimeric RNA/DNA oligonucleotides, as described in Beetham et al, Proc. Natl Acad. Sci. USA 96:8774-8778(1999) and Zhu et al, loc. cit. 8768-8773, or the so-called "library of recombinant gene oligonucleotides" (PCT application WO 03/013226). Similarly, genetically engineered plants or plant cells can be produced by introducing a modified virus, thereby causing a genetic modification in a host, similar to the genetic modification produced in a transgenic plant as described herein, see, for example, U.S. Pat. No. 4,407,956, which describes the present invention. Alternatively, the genetically engineered plant or plant cell may be the product of any natural pathway (i.e., not involving exogenous nucleotide sequences) by introducing nucleic acid sequences derived solely from the host plant species or from sexually suitable plant species. See, for example, U.S. published application No. 2004/0107455.

A genomic library is a collection of clones that contain at least one copy of the major single DNA sequence in the genome.

The NBBl sequence was identified on a cDNA chip prepared from a cDNA library of Nicotiana sylvestris (Nicotiana sylvestris) according to the protocol of Katoh et al (Proc. Japan acad.,79(Ser.B):151-154 (2003)). NBBl is also regulated by nicotine biosynthesis regulatory sites, NICl and NIC 2. NBBl and PMT have the same expression pattern in tobacco plants. NBB1 was implicated in nicotine biosynthesis as evidenced by the fact that, like PMT and a622, NBBl is under NIC gene regulation and shows similar expression patterns.

NBBl expression refers to the biosynthesis of the gene product encoded by SEQ ID NO 3. NBBl inhibition refers to a decrease in NBBl expression. NBBl inhibition has the ability to down-regulate nicotine alkaloid content.

The NICl and NIC2 sites are two independent gene sites in tobacco (n.tabacum), previously designated a and B. Mutant nic and nic2 reduced the expression level of nicotine biosynthetic enzymes and nicotine content, typically nicotine content wild type > homozygous nic2> homozygous nic and homozygous nic2 plants. Legg and Collins, Can.J.Cyto.13:287 (1971); hibi et al, Plant Cell 6:723-735 (1994); reed and Jelesko, Plant Science 167:1123 (2004).

Nicotine is the major alkaloid in tobacco and other species of the nicotiana genus. Other plants have nicotine-producing capabilities and include, for example, the genera solanum (Duboisia), anthocercici, and salpiglossis in the solanaceae family, and the genera Eclipta (Eclipta) and Zinnia (Zinnia) in the Compositae family.

A plant is a term that includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, and plant cells, as well as progeny of the same. Plant material includes, but is not limited to, seed suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots and seedlings, gametophytes, sporophytes, pollen, and microspores. The species of plants used in the present invention are generally as broad as the species of higher plants that can be adapted to transformation techniques, including monocotyledonous and dicotyledonous plants. Preferred plants are plants of the genera Nicotiana, Solanum, Anthocericis and Salpiglossis in the family Solanaceae, or plants of the genera Ecliptae and zinnia in the family Compositae that have nicotine-producing ability. A particularly preferred plant is tobacco (Nicotiana tabacum).

Protein refers to a polymer of amino acid residues.

Reduced nicotine plants include genetically engineered plants having less than half, preferably less than 25%, and more preferably less than 20% or less than 10% nicotine content of a non-transgenic control plant of the same type. Reduced nicotine plants also include genetically engineered plants that have less total alkaloids than the control plants.

A structural gene refers to a sequence of DNA that is transcribed into messenger RNA (mrna) that is subsequently translated into a particular polypeptide characterized by an amino acid sequence. "messenger RNA (mRNA)" means an RNA molecule that contains information encoding the amino acid sequence of a protein.

Sequence identity or "identity" of two nucleic acid or polypeptide sequences herein includes the maximum identity when aligned with reference to the identical residues in the specified regions of the two sequences. When percentage of sequence identity is used for protein reference, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where the amino acid residue is substituted with other amino acid residues of similar chemical nature (e.g., charge and hydrophobicity), and therefore do not alter the functionality of the molecule. Where there is a difference in conservative substitutions, the percent sequence identity may be corrected to a high value due to the conservative nature of the substitution. Sequences that differ by conservative substitutions are said to have "sequence similarity" or "similarity". Means for such modification are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as part rather than a complete mismatch, thus increasing the percentage of sequence identity. Thus, for example, an identical amino acid is scored as 1 and a non-conservative substitution is scored as zero, with conservative substitutions being scored between zero and 1. For example, the score for conservative substitutions is calculated according to the algorithm of Meyers and Miller. Computer applied biol. Sci.4:11-17(1988), similarly implemented using the PC/GENE program (Intelligenetics, Mountain View, California, USA).

Percentage sequence identity as used herein means a characteristic value (determined by optimal alignment of two sequences over a comparison window), wherein the portion of the polynucleotide sequence in the comparison window may include insertions or deletions (i.e., gaps) as compared to a reference sequence (which does not include insertions or deletions) for the purpose of optimal alignment of the two sequences. The percentages are calculated by: the number of positions of the identical nucleic acid base or amino acid residue in both sequences is determined to yield the number of positions that can be matched, the number of matched positions is divided by the total number of positions in the window of alignment, and the result is multiplied by 100 to yield the percentage of sequence identity.

Sequence identity has a meaning recognized in the art and can be calculated using published techniques. See, for example, COMPATIONAL MOLECULAR BIOLOGY, Lesk Master (Oxford University Press, 1988), Biocumputing: INFORMATICS AND GENOME PROJECTS, Smith Master (Academic Press,1993) COMPUTER ANALYSIS OF SEQUENCE DATA, part I, Griffin AND Griffin Master (Humana Press,1994), SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, Von Heinje Master, Academic Press (1987), SEQUENCE ANALYSIS PRIMER, Gribskov AND Deveux master (Macmillan StocktonPress,1991), AND Carllo AND Lipton, SIAMJ.applied Math.48:1073 (1988). Methods commonly used To determine identity or similarity between two sequences include, but are not limited To, those disclosed in GUIDE TO HUGE COMPOSITS (Bishop eds., Academic Press,1994), and Carillo and Lipton, supra. Methods of determining identity and similarity are written as 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 et al, J.MoL biol.215:403(1990)), and FASTDB (Brutlag et al, Comp.App.biosci.6:237 (1990)).

Tobacco refers to any plant of the genus nicotiana that produces nicotine alkaloids. Tobacco also includes material products produced by plants of the genus nicotiana, including, for example, cigarettes, cigars, chewing tobacco, snuff and cigarettes produced with genetically engineered low nicotine tobacco for smoking cessation. Examples of tobacco species include, but are not limited to, flower tobacco (n.alata), tabacco (n.glauca), long flower tobacco (n.longiflora), n.persica, yellow flower tobacco (n.rustica), forest tobacco (n.sylvestris), and tobacco (n.tabacum).

Tobacco Specific Nitrosamines (TSNAs) are carcinogens that form significantly during the curing, processing and smoking of tobacco. Hoffman, D, et al, J.Natl Cancer Inst.58,1841-4 (1977); wiernik A et al, RecentrtAdv. Tob. Sci, (1995),21: 39-80. TSNAs, such as 4- (N-nitrosomethylamino) -1- (3-pyridyl) -1-butanone (NNK), N ' -nitrosonornicotine (NNN), N ' -Nitrosoanatabine (NAT), and N ' -Nitrosopseudoanatabine (NAB), are formed by the N-nitrosation of nicotine and other minor amounts of tobacco alkaloids, such as nornicotine, anatabine, and quinaldine. The reduction in nicotine alkaloid levels reduces TSNAs levels in tobacco and tobacco products.

Tobacco hairy roots are those tobacco roots which have T-DNA (from the Ri plasmid of Agrobacterium rhizogenes and which have been integrated into the tobacco genome) and which have been grown in culture without supplementation with auxins and other prohormones. Tobacco hairy roots produce nicotine alkaloids like tobacco plant roots.

By transgenic plant is meant a plant that comprises a nucleic acid sequence that is native to another organism or species or that has been optimized for the nucleic acid sequence from another organism or species due to the codon usage of the host.

Transgenic plants can be produced by any genetic transformation method. For example, suitable transformation methods include Agrobacterium-mediated transformation, particle bombardment, electroporation, polyethylene glycol fusion, transposon tagging, and site-directed mutagenesis. The identification and selection of transgenic plants is a well-known technique, and the detailed technique thereof need not be repeated here.

A variant is a nucleotide or amino acid sequence that differs from a standard or established sequence of nucleotide or amino acid sequences of a particular gene or protein. The terms "isoform (isoform)", "isoform (isotype)" and "analog (analog)" also refer to "variant" forms of a nucleotide or amino acid sequence. An amino acid sequence that is altered by the insertion, deletion or substitution of one or more amino acids, or that is altered by the nucleotide sequence, can be considered a "variant" sequence. The variant may have "conservative" changes, where the substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. Variants may also have "non-conservative" changes, such as replacement of glycine with tryptophan. Minor variants of the same function may also include amino acid deletions or insertions, or both. It has been found that the determination of which amino acid residues can be substituted, inserted, or deleted can be guided by computer programs well known in the art, such as Vector NTISuite (InforMax, MD) software. "variant" may also refer to "shuffled gene" as described in patents assigned to Maxygen.

The invention is not limited to the particular methodology, protocols, vectors, and reagents, etc., described herein. These may be modified. Furthermore, the terminology referred to above in this description is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, the parameter "a gene" is one or more genes and includes equivalent amounts known to the skilled artisan, and so forth.

Polynucleotide sequences

Nicotine alkaloid biosynthesis genes have been identified in several plant species, such as plants of the nicotiana genus. Accordingly, the invention encompasses any nucleic acid, gene, polynucleotide, DNA, RNA, mRNA or cDNA molecule that is isolated from the genome of a plant species and that down-regulates nicotine alkaloid biosynthesis.

For example, inhibition of at least one of a622 and NBBl may down-regulate nicotine levels in a plant. In addition, nicotine alkaloid levels, such as at least one of QPT and PMT, and at least one of a622 and NBBl, can be further reduced by inhibiting the expression of nicotine biosynthesis genes. The polygene-repressed plant may be obtained by plant regeneration from a polygene-repressed genetically engineered plant cell or by crossing a nicotine biosynthesis gene-repressed genetically engineered first plant with a nicotine biosynthesis gene-repressed genetically engineered second plant.

In one aspect, the invention provides isolated nucleic acid molecules comprising SEQ ID NO 1; a polynucleotide sequence encoding the polypeptide of SEQ ID NO. 2; 1 and encoding a622 polypeptide; and polynucleotide sequences which differ from SEQ ID NO.1 due to the degeneracy of the genetic code. Further aspects of the invention are the peptide encoded by SEQ ID NO.1 and said SEQ ID NO. 2.

In another aspect, the invention provides isolated nucleic acid molecules comprising SEQ ID NO 3; a polynucleotide sequence encoding the polypeptide of SEQ ID NO. 4; a polynucleotide sequence that hybridizes to SEQ ID NO.3 and encodes an NBB1 polypeptide; and a polynucleotide sequence which differs from SEQ ID NO.3 due to the degeneracy of the genetic code. Further aspects of the invention are the peptide encoded by SEQ ID NO.3 and said SEQ ID NO. 4.

The invention further provides a nucleic acid which is complementary to SEQ ID NO.1 or SEQ ID NO.3 and also provides a nucleic acid comprising at least 15 contiguous bases which hybridizes to SEQ ID NO.1 or SEQ ID NO.3 under medium or high stringency conditions as described below. For the purposes of the present description, the category of nucleic acids hybridizing to SEQ ID NO.3 excludes nucleic acids having the sequence SEQ ID NO. 559 as disclosed in International application WO 03/097790.

In a further embodiment, the siRNA molecule of the present invention comprises a polynucleotide that inhibits the expression of either SEQ ID No.1 or 3, although the sequence of SEQ ID No.1 or SEQ ID No.3 is not limited. The siRNA molecules of the invention may include any sequence that is linked to a622 or NBBl, for example, about 15 to about 25 or more, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or more linked nucleotides. In this context, the term siRNA molecule also encompasses molecules having the nucleotide sequence SEQ ID NO:559 described in the above-mentioned WO 03/097790, but also any fragments thereof.

An "isolated" nucleic acid molecule is a nucleic acid molecule of interest, DNA or RNA, which has been removed from its endogenous environment. For example, for the purposes of the present invention, a recombinant DNA molecule contained within a DNA construct is considered to be isolated. Still further embodiments of isolated DNA molecules include recombinant DNA molecules maintained in a heterologous host cell or DNA molecules that have been purified, partially or substantially purified in solution. Isolating RNA molecules includes in vitro RNA transcripts of the DNA molecules of the invention. According to the present invention, isolated nucleic acid molecules further include synthetically produced molecules.

The nucleic acid molecules of the invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for example, cDNA and genomic DNA produced by cloning or synthesis. The DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA can be the coding strand, also known as the sense strand, or the non-coding strand, also known as the antisense strand.

Unless otherwise stated, all nucleotide sequences determined by sequencing DNA molecules herein were determined using an automated DNA sequencer (e.g., model 373 from Applied Biosystems, Inc.). Thus, as is known in the art, any DNA sequence determined by such automated methods may contain some error in any nucleotide sequence determined herein. Typically, the nucleotide sequence determined by automation is at least about 95% identical, more typically at least about 96% to at least about 99.9% identical, to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more accurately determined by other means, including manual DNA sequencing methods well known in the art. As is well known in the art, a single insertion or deletion within a determined nucleotide sequence as compared to the actual sequence of the nucleotide will result in a frame shift mutation (frame shift) in the translation of the nucleotide sequence such that the determined nucleotide sequence encodes a predicted amino acid sequence that is completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the site of the insertion or deletion.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide as described above that hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention. A polynucleotide that hybridizes to a "portion" of a polynucleotide means a polynucleotide, DNA or RNA, that hybridizes to at least about 15 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least about 30 nucleotides, and more preferably more than 30 nucleotides of a reference polynucleotide

For the purposes of the present invention, two sequences hybridize when they form a double-stranded complex in a hybridization solution (6 XSSC, 0.5% SDS, 5 XDenhardt's solution and 100. mu.g of nonspecific carrier DNA). See Ausubel et al, supra, section 2.9, appendix 27 (1994). Sequences may hybridize under "moderately stringent" conditions, which are: 6 XSSC, 0.5% SDS, 5 XDenhardt's solution and 100. mu.g of non-specific vector DNA at 60 ℃. For hybridization under "high stringency" conditions, the temperature was increased to 68 ℃. After hybridization under moderately stringent conditions, the nucleotides are washed 5 times with a solution of 2 XSSC plus 0.05% SDS at room temperature, followed by a wash with a solution of 0.1 XSSC plus 0.1% SDS at 60 ℃ for 1 hour. For high stringency conditions, the wash temperature was increased to 68 ℃. For the purposes of the present invention, hybridized nucleotides are those detected with 1ng of a radiolabeled probe having a radioactive specificity of 10,000cpm/ng, which is clearly visible to the naked eye after exposure to X-rays at 70 ℃ for up to 72 hours.

Nucleic acid molecules which are at least 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences SEQ ID NO 1 or 3 are referred to herein. Preferred nucleic acid molecules are at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences SEQ ID NO 1 or 3 shown. The two nucleic acid sequences may differ at the 5 'or 3' end position of the reference nucleotide sequence or at any position between the two terminal positions, either individually dispersed among nucleotides within the reference sequence or dispersed within one or more linking groups within the reference sequence.

As a special case, whether any particular nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to a reference nucleotide sequence means that the alignment is performed using standard algorithms well known in the art and can generally be determined using well known computer programs such as the BLASTN algorithm. See Altschul et al, Nucleic Acids Res.25:3389-3402 (1997).

In order to produce an RNA product that is complementary to the entire A622 or NBBl mRNA sequence or a portion thereof, the antisense methods of the invention may employ heterologous sequences. This sequence may be complementary to any contiguous sequence of the natural messenger RNA, that is, it may be complementary to the endogenous mRNA sequence proximal to the 5' end or the capping site (the capping site), downstream of the capping site, between the capping site and the start codon, and may cover all or only a portion of the non-coding region, may be linked to the non-coding region and the coding region, complementary to all or part of the coding region, complementary to the 3' end of the coding region, or complementary to the 3' -untranslated region of the mRNA.

Suitable antisense sequences can be 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 sequence may be extended or shortened at its 3 'or 5' end.

The particular antisense sequence and the length of the antisense sequence can vary, for example, depending on the desired degree of inhibition and the stability of the antisense sequence. The generally available techniques and information provided herein can guide the selection of an appropriate a622 or NBBl antisense sequence. As used herein, with respect to SEQ ID NO:1 or 3, the oligonucleotide of the invention may be a contiguous segment of the cDNA sequence of A622 or NBBl in antisense orientation of any length sufficient to achieve the desired effect when transformed into a recipient plant cell.

The present invention contemplates sense co-suppression of one or both of A622 and NBBl. When expressed in a plant cell, the sense polynucleotide used in the practice of the present invention has a length sufficient to inhibit the natural expression of plant A622 or NBBl protein in the plant cell. Such sense polynucleotide may be the entire genomic nucleic acid or a complementary nucleic acid encoding the a622 or NBBl enzyme, or a fragment thereof, such fragment typically being at least 15 nucleotides in length. Techniques that can be used to determine the length of sense DNA that inhibits expression of an endogenous gene in a cell are common.

In one embodiment of the invention, plant cells are transformed with a nucleic acid construct comprising a polynucleotide fragment encoding an enzymatic RNA molecule ("ribozyme") that directly destroys (i.e., cleaves) the mRNA transcript of DNA encoding a622 or NBBl, as described herein. Ribozymes contain a substrate binding region that binds to an accessible region of a target mRNA and which catalyzes the breakdown of ribonucleic acids, preventing translation and protein production. The binding region may include an antisense sequence complementary to a target mRNA sequence; the catalytic site may be a hammerhead site or other site, such as a hairpin site.

Ribozyme cleavage sites within an RNA target can be initially identified by scanning the target molecule to identify ribozyme cleavage sites (e.g., GUA, GUU, or GUC sequences). Once identified, short RNA sequences of 15, 20, 30, or more ribonucleotides of the corresponding region of the target gene (containing the cleavage site) can be evaluated for predicting structural features.

The suitability of candidate targets can also be assessed by testing their accessibility (accessibility) to hybridization with complementary oligonucleotides using ribonuclease protection assays known in the art. The DNA encoding the enzymatic ribonucleic acid molecule can be produced according to known techniques. See, for example, Cecil et al, U.S. patent No. 4,987,071; keene et al, U.S. Pat. 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. Pat. 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 U.S. patent No. 5,622,854.

The production of such enzymatic ribonucleic acid molecules and cleavage of the A622 or NBBl protein products in plant cells reduces the protein activity in plant cells essentially in the same way as an antisense RNA molecule is produced; that is, by interfering with the translation of the mRNA in the cell that produces the enzyme. The term "ribozyme" describes an RNA-containing nucleic acid that functions as an enzyme, such as an endoribonuclease, and may be used interchangeably with "enzymatic RNA molecule".

The invention further includes nucleic acids encoding ribozymes, nucleic acids encoding ribozymes and which have been inserted into expression vectors, host cells containing such vectors, and methods of using ribozymes to reduce A622 and NBBl expression in plants.

In one embodiment, the invention provides a double-stranded nucleic acid molecule that mediates RNA interference gene silencing (mediate rnalnterference gene silencing). In another embodiment, the siRNA molecules of the invention are comprised of double-stranded nucleic acid molecules comprising about 15 to about 30 base pairs between oligonucleotides, which comprise 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 yet another embodiment, siNA molecules of the invention include double-stranded nucleic acid molecules having about 1 to about 32 (e.g., about 1, 2, or 3) nucleotide overhanging ends (overhanging ends), e.g., about 21 nucleotide duplexes having about 19 base pairs and 3' terminal single, di, or trinucleotide overhangs (overhanging). In yet another embodiment, siNA molecules of the invention comprise double stranded nucleic acid molecules having blunt ends (blunt ends), wherein both ends are blunt, or one of the two ends is selected to be blunt.

The siNA molecules of the invention may include modified nucleotides that still retain the ability to mediate RNAi. Modified nucleotides can be used to improve in vivo or in vitro properties such as stability, activity, and/or bioavailability. For example, siNA molecules of the invention may comprise modified nucleotides in amounts expressed as a percentage of the total number of nucleotides in the siNA molecule. Likewise, siNA molecules of the invention may typically comprise 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). For example, the actual percentage of modified nucleotides in a given siNA molecule depends on the total number of nucleotides in the siNA. If the siNA molecule is single-stranded, the percentage of modification may be based on the total number of nucleotides in the single-stranded siNA molecule. Likewise, if the siNA molecule is double-stranded, the percentage of modification may be based on the total number of nucleotides in the sense strand, the antisense strand, or both.

For example, expression of a622 and NBBl can be reduced by genetic engineering methods well known in the art. Expression may be reduced by introducing a nucleic acid construct capable of causing expression of an RNA comprising a partial sequence encoding A622 or NBBl. The partial sequence may be in the sense or antisense orientation. The partial sequence may be present in an inverted repeat sequence capable of forming a double stranded RNA region. Expression may be reduced by introducing a nucleic acid construct encoding an enzymatic RNA molecule (i.e., a "ribozyme") that directly destroys (i.e., cleaves) the niRNA transcript of the DNA encoding a622 or NBBl. Expression may be reduced by introducing a nucleic acid comprising a622 or NBBl part sequence which causes targeted in situ mutation (targeted initu mutagenesis) of the endogenous gene, resulting in its inactivation.

Sequence analysis

Methods of calculating sequence alignments are well known in the art. The optimal algorithm for sequence alignment can be performed according to the local homology algorithm of Smith and Waterman. adv.appl.Math.2:482 (1981); homology alignment by Needleman and Wunsch, J.MoI.biol.48:443 (1970); methods for similarity search by Pearson and Lipman, proc.natl.acad.sci.usa 85:2444 (1988); by computerisation of these algorithms, including but not limited to CLUSTAL, Intelligenetics, Mountain View, California in PC/Gene programs; GAP, BESTFIT, BLAST FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group (GCG), 575Science Dr., Madison, Wisconsin, USA; the CLUSTAL program is described in detail in: higgins and Sharp, Gene 73: 237-; higgins and Sharp, CABIOS 5: 151-; 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-.

A family of BLAST programs that can be used for database similarity searches includes BLASTN for alignment of nucleotide query sequences to nucleotide database sequences; BLASTX is used for alignment of nucleotide query sequences with protein database sequences; BLASTP is used for alignment of protein query sequences to protein database sequences; TBLASTN is used for alignment of protein query sequences with nucleotide database sequences; and TBLASTX for alignment of nucleotide query sequences to nucleotide database sequences. See, Current Protocols in Molecular Biology, chapter 19, eds. Ausubel et al, Greene publishing and Wiley-Interscience, New York (1995); altschul et al, MoI.biol.215: 403-; and, Altschul et al, Nucleic Acids Res.25:3389-3402 (1997).

Software for performing BLAST analysis is common, for example, by the national Center for Biotechnology Information. These algorithms identify high scoring sequence pairs (HSPs) for the first time by identifying short bytes of length W in the query sequence, which either match or satisfy some positive-valued threshold T when identical to a byte of the same length in a database sequence. T refers to the threshold of the adjacent byte. These initial adjacent byte sample numbers act as seeds to initiate searches for longer HSPs containing them. The byte sample number is extended in both directions of each sequence in order to maximize the incremental alignment score. Increasing scores for nucleotide sequences were calculated using the parameters M (positive assignment of matched residue pairs; always >0) and N (negative assignment of mismatched residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the incremental score. The extension of the number of byte samples in each direction stops when: increasing alignment score by an amount X back off its maximum value reached; increasing scores back to 0 or below 0 as one or more negatively scored residue alignments accumulate; or to the end of the sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. Default values for the byte length (W) of the BLASTN program (for nucleotide sequences) are 11, the expectation (E) is 10, cutoff is 100, M-5, N-4, and two strand alignment. The default value for the byte length (W) of the BLASTP program for amino acid sequences is3, the expectation value (E) is 10, and the BLOSUM62 score matrix. See Henikoff and Henikoff, Proc. Natl. Acad. ScI USA 89:10915 (1998).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity of two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873- > 5877 (1993)). The measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides a representation of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

Sequence multiple alignments can be performed using the CLUSTAL alignment method (Higgins and Sharp, CABIOS 5:151-153(1989)) with default parameters GAP PENALTY ═ 1O and GAP LENGTH PENALTY ═ 1O). The default parameters for pairwise alignment using CLUSTAL method are KTUPLE 1, GAP PENALTY ═ 3, WINDOW ═ 5 and DIAGONALS SAVED ═ 5.

The following working parameters are preferred for determining alignments and similarities using BLASTN, which contribute to the percent identity and E value of polynucleotide sequences Unix run commands blastall-p BLASTN-d embldb-E10-G0-EO-r 1-v 30-b 30-i queryseq-o results; the parameter is: -p program name [ string ]; -d database [ string ]; -E desired value (E) [ real ]; -G open gap value (default 0) [ integer ]; -r forward assignments of nucleotide matches (blastn only) [ integer ]; -V a line specification code (V) [ integer ]; -B display codes of alignment (B) [ integer ]; -i query the file [ incoming file ]; and-o output BLAST report file as you go [ file exit ].

"number of samples" is obtained by searching one or more database sequences generated by BLASTN, FASTA BLASTP, or similar algorithms against a sequence, aligning and identifying similar portions of the sequence. The number of samples is sorted in order of similarity and sequence coverage length. The number of samples for a database sequence typically means that only a portion of the query sequence length is covered.

The BLASTN, FASTA and BLASTP algorithms also produce aligned "expect" values. The expectation value (E) shows the number of samples that one can "expect" to have a chance to see a certain number of consecutive sequences when one searches a database of a certain size. This expected value is used as a threshold for determining significance, which determines whether a sample to a database, such as the preferred EMBL database, shows true similarity. For example, a polynucleotide sample with an E value of 0.1 is interpreted to mean that in a database of the same size as the EMBL database, one would expect to have an opportunity to see a 0.1 match for the aligned portion of the sequence using only a simple similarity scoring method. Using this index, the probability that the aligned and matched portions of the polynucleotide sequences are identical is 90%. Using the BLASTN or FASTA algorithm, there is a 1% or less chance that a match will be found in the EMBL database for aligned and matched portions of the sequence having an E value of 0.01 or less.

According to a specific embodiment, with respect to each polynucleotide of the present invention, a "variant" polynucleotide preferably includes a sequence having the same or fewer number of nucleic acids per polynucleotide of the present invention and exhibiting an E-value of 0.01 or less as compared to the polynucleotide of the present invention. That is, a variant polynucleotide is any sequence that has a probability of being at least 99% identical to a polynucleotide of the invention, as measured by the BLASTN, FASTA, or BLASTP algorithm using the setting parameters described above, with an E value of 0.01 or less. Alternatively, variant polynucleotides of the invention hybridize under stringent conditions to the polynucleotide sequences SEQ ID NO 1 or 3, or to complementary, reverse sequences, or to the reverse complement of those sequences.

The present invention also includes polynucleotides that differ from those disclosed, except as a result of the degeneracy of the genetic code, which encodes the same polypeptide as that encoded by a polynucleotide of the present invention. Thus, where the polynucleotide includes a sequence other than the polynucleotide sequence of SEQ ID NO 1 or 3, the reverse sequence thereof, or the reverse complement thereof, the results of conservative substitutions are contemplated by and included within the present invention. In addition, sequences other than the polynucleotide sequence SEQ ID NO 1 or 3, the reverse sequence thereof, or the reverse complement thereof, the sum of deletions and/or insertions which is less than 10% of the total length of the sequence are contemplated by and are included in the present invention.

In addition to having a specified percentage of identity with a polynucleotide sequence of the invention, variant polynucleotides preferably have structure and/or functionality other than that of a polynucleotide of the invention. In addition to having a high degree of similarity in primary structure to the polynucleotides of the invention, which have a specified degree of identity or are capable of hybridizing, preferably at least one of the features (i) they comprise an open reading frame or partial open reading frame, encoding a polypeptide having substantially the same functionality as the polypeptide encoded by the polynucleotide of the invention; or (ii) they have a common region. For example, a variant polynucleotide may encode a polypeptide capable of inhibiting a622 or NBBl.

Nucleic acid constructs

According to one aspect of the invention, a sequence that reduces nicotine alkaloid biosynthesis is incorporated into a nucleic acid construct suitable for plant transformation. For example, such a nucleic acid construct may be used to reduce gene expression of at least one of a622 or NBBl in a plant. In addition, the nucleic acid construct of the invention can reduce the expression of one or both of a622 and NBBl and the polynucleotide sequence encodes a nicotine biosynthetic enzyme.

Accordingly, nucleic acid constructs are provided comprising a sequence that down-regulates nicotine alkaloid biosynthesis under the operable control of a transcription initiation region in a plant such that the construct is capable of producing RNA in a host plant cell.

Recombinant DNA constructs can be prepared using standard techniques. For example, the DNA sequence for transcription can be obtained by excising an appropriate fragment by treating a vector containing the sequence with a restriction enzyme. The DNA sequence for transcription can also be generated by annealing and ligating synthetic oligonucleotides or by Polymerase Chain Reaction (PCR) using synthetic oligonucleotides to generate appropriate restriction sites at each end. The DNA sequence is then cloned into a vector containing suitable regulatory elements, such as an upstream promoter and a downstream terminator sequence.

Suitable regulatory elements

Promoter means the region of DNA upstream of the transcription initiation site involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "constitutive promoter" is a promoter that is active throughout the life of a plant and under most environmental conditions. Tissue-specific, tissue-preferred, cell-specific, and inducible promoters constitute such "non-constitutive promoters". "operably linked" refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. In general, "operably linked" means that the nucleic acid sequences being linked are contiguous.

The promoter used to reduce expression of the nucleic acid sequence introduced into the cell to reduce expression of A622 or NBBl may be a constitutive promoter or a tissue-specific, tissue-preferred, cell-specific, and inducible promoter. Preferred promoters include promoters active in root tissue, such as the tobacco RB7 promoter (Hsu et al, pesticide, Sci.44:9-19 (1995); U.S. Pat. No. 5,459,252) and promoters active under conditions capable of producing enhanced expression of enzymes involved in nicotine biosynthesis, such as the tobacco RD2 promoter (U.S. Pat. No. 5,837,876), the PMT promoter (Shoji et al, Plant CellPhysiol,41:831-839(2000 b); WO 2002/038588) or the A622 promoter (Shoji et al, Plant MoI Biol,50:427-440 (2002)).

The vectors of the invention may also comprise a termination sequence located downstream of the nucleic acid molecules of the invention so as to terminate transcription of mRNA and increase in polyA sequences. Such exemplary terminators are the cauliflower mosaic virus (CaMV)35S terminator and the nopaline synthase gene (Tnos) terminator. The expression vector may also contain an enhancer, an initiation codon, a splicing signal sequence, and a target sequence.

The expression vectors of the invention may also comprise a selectable marker (whereby transformed plant cells are identified during culture). The marker may be linked to a heterologous nucleic acid molecule, i.e., the gene is operably linked to a promoter. As used herein, the term "marker" refers to a gene encoding a trait or a phenotype that allows for the selection or screening of plants or plant cells containing the marker. Typically, the marker gene encodes for antibiotic or herbicide resistance. This allows selection of transformed cells from untransformed or transfected cells.

Examples of suitable selectable markers include adenosine deaminase, dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidine kinase, xanthine-guanine phosphoribosyltransferase, glyphosate and glufosinate resistance, and amino-glycoside 3' -O-phosphotransferase (kanamycin, neomycin and G418 resistance). These markers include resistance to G418, hygromycin, bleomycin, kanamycin and gentamicin. The construct may also comprise a selectable marker gene, Bar, which confers resistance to the herbicide glufosinate analogue, such as ammonium gluphosinate. Thompson et al, EMBO J.9:2519-2523 (1987). Other suitable selectable markers are well known.

A macroscopic marker such as Green Fluorescent Protein (GFP) can be used. Methods for identifying transformed plants based on controls for 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, allowing the vector to be cloned into a bacterial or phage host. Preferably, a wide host range of prokaryotic origin replication sequences is used. Selectable markers for bacteria may be included for selection of bacterial cells carrying the desired construct. Suitable prokaryotic selectable markers also include resistance to antibiotics such as kanamycin tetracycline.

Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art. For example, when Agrobacterium is the host, T-DNA sequences may be included to facilitate subsequent transfer and integration into the plant chromosome.

Plants for genetic engineering

The invention includes genetic manipulation of plants to inhibit nicotine alkaloid synthesis by introducing a polynucleotide sequence that down-regulates the expression of a gene that regulates nicotine alkaloid synthesis, such as A622 and NBBl. The result is a plant with reduced nicotine alkaloid levels.

In this specification, "plant" means any plant material containing a nicotine alkaloid which can be genetically manipulated, including but not limited to differentiated or undifferentiated plant cells, gene bodies, whole plants, plant tissues or plant organs, or any part of a plant such as leaves, stems, roots, shoots, tubers, fruits, rhizomes or the like.

Exemplary plants that may be genetically engineered according to the present invention include, but are not limited to, tobacco, potato, tomato, eggplant, green pepper, and belladonna.

Plant transformation and selection

According to the present invention, the constructs can be used to transform any plant cell using suitable transformation techniques. Plant cells of both monocotyledonous and dicotyledonous angiosperms or gymnosperms can be transformed in different ways known in the art. See, for example, Klein et al, Biotechnology 4: 583-; bechtold et al, C.R.Acad.ScL Paris316:1194-1199 (1993); bent et al, MoI.Gen.Genet.204:383-396 (1986); paszowski et al, EMBOJ.3:2717-2722 (1984); sagi et al, Plant Cell Rep.13:262-266 (1994). Agrobacterium tumefaciens species such as Agrobacterium tumefaciens and Agrobacterium rhizogenes can be used, for example, according to Nagel et al, Microbiol Lett 61:325 (1990). Alternatively, plants may be transformed with Agrobacterium rhizogenes (Rhizobium), Rhizobium (Sinorhizobium) or Rhizobium (Mesorhizobium). Broothaerts et al, Nature 433: 629-.

For example, Agrobacterium tumefaciens can be transformed with a plant expression vector, e.g., by electroporation, followed by introduction of the Agrobacterium tumefaciens into a plant cell, e.g., by the well-known leaf disc method. Other methods for achieving this include, but are not limited to, transformation of germinated pollen grains by electroporation, particle gun bombardment, calcium phosphate precipitation and polyethylene glycol fusion, direct transformation (Lorz et al, MoI. Genet.199:179-182(1985)), and other methods known in the art. If a selection marker, such as kanamycin resistance, is used, it is easy to determine which cells have been successfully transformed.

The Agrobacterium tumefaciens transformation methods discussed above are known to be useful for transforming dicotyledonous plants. In addition, cereal monocotyledons have been transformed with Agrobacterium tumefaciens by deIa Pena et al, Nature 325:274-276(1987), Rhodes et al, Science 240:204-207(1988), and Shimamato et al, Nature 328:274-276 (1989). See also Bechtold et al, cr.acad.sci.paris 316(1994), which describes the use of vacuum infiltration for agrobacterium-mediated transformation.

For the purposes of this specification, a plant or plant cell may be transformed with a plasmid comprising one or more sequences, each operably linked to a promoter. For example, an exemplary vector may comprise a QPT sequence operably linked to a promoter. Likewise, the plasmid may comprise a QPT sequence and a622 sequence operably linked to a promoter. Alternatively, plants or plant cells may be transformed with more than one plasmid. For example, a plant or plant cell may be transformed with a first plasmid comprising a promoter operably linked to a plasmid different from a second plasmid comprising the a622 or NBBl sequences. Of course, the first and second plasmids or parts thereof are introduced into the same plant cell.

The genetically engineered plants of the invention may be produced by conventional breeding. For example, a genetically engineered plant with down-regulated QPT and a622 activity may be produced by crossing a transgenic plant having reduced QPT expression with a transgenic plant having reduced a622 expression. Following successive rounds of crossing and selection, genetically engineered plants can be selected that have down-regulated QPT and a622 activity.

The presence of a protein, polypeptide, or nucleic acid molecule in a particular cell can be used to determine, for example, whether a cell has been successfully transformed or transfected.

Pairs of recombination sites that can be recognized by specific recombinases (e.g., ere or flp) can be included in the marker gene to facilitate removal of the marker after selection. See U.S. published application No. 2004/0143874.

The generation of transgenic plants without a marker gene may use a second plasmid comprising a nucleic acid encoding the marker, which is different from the first plasmid comprising the a622 or NBBl sequence. The first and second plasmids or parts thereof are introduced into the same plant cell, such selectable marker gene expression is transient, transformed plant cells are identified, and in the resulting transformed plant the A622 or NBBl sequence is stably integrated into the genome and the selectable marker gene is not stably integrated. See U.S. published application No. 2003/0221213. The first plasmid comprising the a622 or NBBl sequence may optionally be a binary vector with a T-DNA region consisting of a nucleic acid sequence in wild-type non-transgenic tobacco or in a sex-compatible nicotiana species.

The nucleic acid construct of the invention can be used to transform plant cells without the use of a selectable marker or a visible marker, and the regenerated plant can be identified for the transgene by detecting the presence or absence of the introduced construct using PCR or other specific nucleic acid sequence detection methods. The identification of transformed plant cells is simplified by identifying differences in the growth rate or morphological characteristics of the transformed plant cells and the growth rate or morphological characteristics of non-transformed plant cells under similar culture conditions (see WO 2004/076625).

The method of regenerating a transgenic plant from a transformed cell or culture varies depending on the plant species according to known methods. For example, methods for regenerating transgenic tobacco plants are well known.

For the purposes of this specification, genetically engineered plants having down-regulated expression of at least one of a622 and NBBl are selected. In addition, the genetically engineered plants of the invention may have down-regulated expression of nicotine biosynthesis genes, such as QPT or PMT, and at least one of a622 and NBBl.

Nicotine acts as a natural pesticide that helps protect tobacco plants from pests, to which conventionally bred or transgenic low nicotine tobacco has been reported to have increased sensitivity. Legg, p.d., et al, can.j.cyto.,13:287-291 (1971); voelckel C, et al, Chemology 11: 121-; steppuhn, A., et al, PLoBiol, 2(8): e217: 1074-. Thus, it would be desirable to additionally transform low nicotine plants produced by this method with a transgene that confers additional insect protection, such as a gene encoding a Bt insecticidal protein, a protease inhibitor, or a vitamin H binding protein. Introduction of a transgene conferring additional insect protection can be achieved by crossing a transgenic low nicotine plant with a second transgenic plant containing a gene encoding an insect resistance protein.

Quantification of nicotine alkaloid content

The transgenic plants of the invention are characterized by reduced nicotine alkaloid content. In genetically engineered plants, reduction of nicotine alkaloid content is preferably achieved by reducing expression of genes of the nicotine biosynthetic pathway, such as a622 or NBBl.

In plants according to the invention, the phrase "reduced nicotine or nicotine alkaloid content" refers to a quantitative reduction in the nicotine alkaloid content of a plant when compared to a non-transformed control plant. The quantitative decline in nicotine alkaloid levels can be quantified by several methods, such as by gas liquid chromatography, high performance liquid chromatography, radioimmunoassay, and enzyme-linked immunosorbent assay. In the present invention, nicotine alkaloid levels are measured by gas liquid chromatography equipped with a capillary column and FID detector, as described by Hibi, N.et al, Plant Physiology 100:826-835 (1992).

Reduced nicotine alkaloid products

The present invention provides a transgenic plant having reduced nicotine alkaloid levels. For example, the present invention contemplates reducing nicotine levels by inhibiting the expression of at least one of a622 and NBBl. Transgenic plants that have inhibited expression of A622 or NBBl and reduced nicotine content are then selected, and various products can be made from such plants.

Since the present invention provides a method for reducing alkaloids, TSNAs can also be reduced because the alkaloid content in tobacco is significantly positively correlated with TSNA accumulation. For example, the significant correlation coefficient between anatabine and NAT is 0.76. Djordjevic et al, J.Agric.food chem.,37:752-756 (1989). TSNAs are carcinogens that form apparently during the curing, processing, and smoking of tobacco. However, in growing tobacco plants or freshly harvested tobacco, TSNAs are present in small amounts. Hecht and Hoffman, J.Natl.cancer Inst.58,1841-4 (1977); wiernik et al, Recentr adv. Tob. Sci,21:39-80 (1995). The addition of an N ═ O functional group to one of the nitrogens of the secondary or tertiary amine can be readily accomplished by nitrosating agents to form nitrosamines containing an organic functional group N — N ═ O. Although they may potentially occur in other nicotine-containing products, such specific carcinogens are found only in tobacco.

In cigarettes, TSNAs are considered the most prominent carcinogens and their carcinogenic properties have been demonstrated. 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 shown to be causative for oral, esophageal, pancreatic and lung cancers (Hecht and Hoffman, IARC sci. In particular, TSNAs have been shown to be a causative agent of the acute rise in malignant adenomas and lung cancer associated with smoking. (Hoffmann et al, crit. Rev. Toxicol.26,199-211 (1996)).

The four TSNAs considered to be the most important and probably carcinogenic to humans in terms of exposure and carcinogenic potential are N ' -nitrosonornicotine (NNN), 4- (N-nitrosomethylamino) -1- (3-pyridinyl) -1-butanone (NNK), N ' -Nitrosoanatabine (NAT), and N ' -Nitrosoanabasine (NAB). A review is given in IARC graphics on the evaluation of the carcinogenic risk of chemical to humans. Lyon (France) VoI 37,205, page 205-208 (1985). These TSNAs are formed by nitrosation of nicotine and minor tobacco alkaloids, including nornicotine, anatabine, and quinaldine.

Levels of alkaloid compounds in the mainstream cigarette of a non-filter cigarette have been reported as follows (in μ g/cigarette): 3000 parts of nicotine: 100-. Mainstream cigarettes in the United states (with or without filters) contain (in ng/cigarette) 9-180ng NNK, 50-500ng NNN,3-25ng NAB and 55-300ng NAT. Hoffmann, et al, Toxicol. environ. health 41:1-52 (1994). Most importantly, these TSNAs levels in non-mainstream cigarettes exceed those in mainstream cigarettes by a factor of 5-10. Hoffmann, et al (1994).

Xie et al (2004) reported that genetically engineered tobacco carriers 21-41, having reduced nicotine content by down-regulating QPT, had total alkaloid levels of about 2300ppm, which was less than 10% of wild-type tobacco. Mainstream cigarettes made with carriers 21-41 had less than 10% NNN, NAT, NAB and NNK compared to levels in standard whole taste cigarettes made with wild type tobacco.

Strategies to reduce TSNAs by reducing the corresponding tobacco alkaloid precursors are the hot spots of current agricultural tobacco research. Siminszky et al, Proc. Nat. Acad. set USA 102(41) 14919-. Therefore, in order to reduce the formation of total TSNAs, it is imperative to reduce the precursor nicotine alkaloids as much as possible by genetic engineering.

Among them, U.S. Pat. Nos. 5,803,081, 6,135,121, 6,805,134, 6,907,887 and 6,959,712 and U.S. published application Nos. 2005/0034365 and 2005/0072047 discuss methods for reducing tobacco-specific nitrosamines (TSNAs).

The reduced nicotine smoking article may be whole tobacco leaf, cut tobacco, shredded tobacco (shredded tobaco), cut tobacco (cut tobaco) and tobacco fractions. Reduced nicotine smoking articles may include cigarette tobacco, cigar tobacco, snuff, chewing tobacco, pipe tobacco, and cigarettes made from reduced nicotine genetically engineered tobacco for smoking cessation.

Reduced nicotine tobacco may also be used to produce reconstituted tobacco (Recon). Recon is produced from tobacco stems and/or smaller leaf particles by a process similar to typical paper production. These processes require processing of the different tobacco portions to be made into Recon and cutting the tobacco into sizes and shapes similar to cut tobacco made from whole tobacco leaves (rag tobaco). These shredded recans are mixed with shredded tobacco and are ready for cigarette manufacture.

In addition to conventional smoking articles, such as cigarette and cigar tobacco, reduced-nicotine tobacco can be used as a protein source, fiber source, ethanol source, and animal feed source. See U.S. published application No. 2002/0197688. For example, nicotine-reduced tobacco can be used as a Rubisco (ribulose diphosphate carboxylase-oxygenase or fraction 1 protein) source because ribulose of tobacco origin can be easily extracted in crystalline form unlike other plants. The ribulose amount of the essential amino acids is equivalent to or exceeds the FAO tentative regimen, except for a slightly lower methionine level. Ershoff B.H., et al, Society for Experimental biology and Medicine 157: 626-; wildman S.G. Photosythesis Research 73: 243-.

In order for biofuels to replace the world's dependence on non-renewable energy sources in large quantities, by-products such as ribulose help pay the cost of producing these renewable energy sources. Greene et al Growing energy, how Biofuels Can End America's oil Dependence; national Resources defensis coursel 2004). Thus, the more nicotine alkaloids are reduced in tobacco, the greater the likelihood of a successful tobacco biomass system.

In order to identify a sequence encoding a nicotine-related enzyme and introduce a target gene to produce a plant transformant, specific examples are described in the following methods. They are merely exemplary and do not limit the invention.

Example 1: preparation of pRNAi-A622 vector for reducing alkaloid content by downregulating A622 expression

As shown in FIG. 2, plasmid pHANNIBAL (see Wesley et al, Plant J.27:581-590(2001)) was modified to produce plasmid pHANNIBAL-X. The SacI restriction site between the ampicillin resistance gene (Amp) and the 35S promoter was removed by SacI cleavage followed by DNA inactivation and ligation. The Multiple Cloning Site (MCS) was modified as follows. The Bam H I restriction site was added to the MCS between the promoter and Pdk intron by inserting a linker (adaptor) between Xhol and EcoRI sites (5' TCGAACGGGATCCCGCCGCTCGAGCGG). The BamHI site was removed from the MCS between intron and terminator by inserting a linker (5' GATCAGCTCTAGAGCCGAGCTCGC) between the BamHI and Xbal sites and a SacI site was inserted.

The plant RNAi binary vector was prepared using pananibal-X using the protocol shown in figure 3, wherein the isolation of specific "sense" and "antisense" fragments was first obtained by adding specific restriction sites to the ends of the gene fragment of interest, and then the sense and antisense fragments were inserted into the modified pananibal-X plasmid in the desired orientation.

Replacement of the GUS coding region in pBI121(Wesley et al, 2001,) with a DNA fragment containing sense and antisense fragments and the inserted Pdk intron produced an RNAi binary vector.

The 814bp-1160bp region of the A622cDNA was used as a region for forming dsRNA (sense strand, antisense strand). PCR was performed using the A622cDNA cloned into pcDNAII as a template and primers with additional bases encoding restriction enzyme sites, and the target DNA fragment was collected and TA was cloned into pGEM-T vector.

The primer sequence is

Sense strand A622F814-Xhol-A622R1160-KpnI

A622F814-Xhol 5'CCGCTCGAGCGGTCAGAGGAAGATATTCTCCA 3'

A622Rl160-KpnI 5'GGGGTACCCCTGGAATAAGACGAAAAATAG 3'

Antisense strand A622F814-Xbal-A622R1160-ClaI

A622F814-XbaI 5'GCTCTAGAGCTCAGAGGAAGATATTCTCCA 3'

A622R1160-ClaI 5'CCATCGATGGTGGAATAAGACGAAAAATAG 3'

Recombination of the modified pHANNIBAL-X was performed starting with the sense strand followed by the antisense strand. The TA clone DNA fragment was cut with the appropriate restriction enzyme, collected, and ligated into pHANNIBAL-X, which had been cut with the same restriction enzyme. The resulting plasmid contained the inverted repeat DNA sequence of the A622 fragment separated by the Pdk intron.

Treatment with BamH I and Sac I, the RNAi region was excised from pHANNIBAL-X containing both sense and antisense strands and ligated to pBI121 from which the GUS coding region had been removed by similar treatment, resulting in binary vector pRNAi-A622 containing a T-DNA fragment containing the nptll selectable marker cassette and the A622RNAi cassette for plant transformation (FIG. 4).

Example 2A 622 inhibition in tobacco BY-2 cells

Although tobacco BY-2 cell cultures are generally unable to synthesize nicotine alkaloids, methyl jasmonate treatment induces gene expression to produce known enzymes of the nicotine biosynthetic pathway and causes the production of nicotine alkaloids.

To deduce the function of A622, RNAi strains were prepared to culture cells in which mRNA from pRNAi-A622 was expressed in cultured tobacco BY-2 cells to inhibit A622.

Agrobacterium transformation (Agrotransformation)

The vector (pRNAi-A622) was transformed into Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain EH105, which was used to transform tobacco BY-2 cells. The method for infecting and selecting tobacco BY-2 cells was as follows.

4ml of BY-2 cells have been cultured for 7 days in 100ml of modified LS medium, see Imanishi et al, Plant mol. biol.,38:1101-1111(1998), passaged to 100ml of modified LS medium for 4 days.

100. mu.l of Agrobacterium tumefaciens solution that had been cultured in YEB medium for 1 day was added to 4ml of cells that had been cultured for 4 days, and the two were co-cultured together in the dark at 27 ℃ for 40 hours.

After cultivation, the cells were washed twice with modified LS medium to remove Agrobacterium

The washed cells were dispersed in a modified LS selection medium containing kanamycin (50mg/l) and carbenicillin (250mg/l) and the cells that had been transformed were selected.

After approximately 2 weeks of culture in the dark at 27 ℃, the transformed cells were transferred to fresh modified LS selection medium and cultured in the dark at 27 ℃ for 1 week.

The transformed cells were then cultured in 30ml of liquid modified LS medium in suspension at 27 ℃ in the dark for 1 week.

1ml of the cultured transformed cells were subcultured in 100ml of modified LS medium. The transformed cells were subcultured every 7 days in the same manner as the wild-type cells.

Alkaloid Synthesis 10ml of each of the 7-day-cultured, transformed BY-2 cells and, as a control, cultured tobacco cells that had been transformed with a Green Fluorescent Protein (GFP) expression vector were washed twice with modified 2, 4-D-free LS medium, and, after adding the 2, 4-D-free modified LS medium to a total of 100ml, suspension-cultured at 27 ℃ for 12 hours.

After adding 100. mu.l of methyl jasmonate (MeJa) diluted to 50. mu.M with DMSO, the cells were cultured in suspension at 27 ℃ for 48 hours.

Jasmonate treated cells were filtered, collected, and freeze-dried. To 50mg of the freeze-dried sample was added 3ml of 0.1N sulfuric acid. The mixture was sonicated for 15 minutes and filtered. To 1ml of the filtrate was added a 28% ammonium (ammonium) solution and centrifuged at 15000rpm for 10 minutes.

1ml of the supernatant was added to an Extrelut-1 column (Merck) and allowed to stand for 5 minutes. Eluted with 6ml of chloroform. The eluate was dried under reduced pressure at 37 ℃ using an evaporator (Taitec Concentrator TC-8).

The dried sample was dissolved in 50. mu.l of 0.1% dodecane in ethanol. The samples were analyzed by gas chromatography equipped with a capillary column (GC-14B) and FID detector. A RESTEC Rtx-5Amine column (Restec) was used as the capillary column. The column temperature was maintained at 100 ℃ for 10 minutes, increased to 150 ℃ at a rate of 25 ℃/min, held at 150 ℃ for 1 minute, increased to 170 ℃ at a rate of 1 ℃/min, held at 170 ℃ for 2 minutes, increased to 300 ℃ at a rate of 30 ℃/min, and then held at 300 ℃ for 10 minutes. The injection and detection temperatures were 300 ℃. Samples of 1. mu.l each were injected and nicotine alkaloid was quantified by the internal standard method.

As shown in fig. 5, jasmonate induction did not produce high accumulation of anatabine (the major alkaloid induced in cultured cells), anatabine, nicotine, or taxinine in the transgenic BY-2 line in which a622 gene expression was inhibited BY RNAi (A3, a21a33, and a43 lines) compared to the control cell line.

RNA expression

To determine whether a decrease in alkaloid accumulation in the a622-RNAi line was specifically associated with a decrease in a622 expression, rather than indirectly affecting expression of known enzyme genes in the nicotine biosynthetic pathway, the levels of a622 and other gene expression were measured in the methyl jasmonate-treated lines, transgenic lines, and control lines at the same time.

Total RNAs were isolated from wild type and transgenic BY-2 cell lines treated with 50. mu.M MeJA for 48 hours. The RNA level of a specific gene was determined by RT-PCR. RNA was extracted using Rneasy plant mini kit (RneasyPlant mini kit) (Qiagen) according to the manufacturer's recommendations. cDNA was synthesized using random hexamers and the Superscript First Strand Synthesis System (Superscript First-Strand Synthesis System) for RT-PCR (Invitrogen). RT-PCR was carried out using TaKaRa Ex Taq enzyme with 5ng of cDNA as template under conditions of 22 cycles for detection of A622, NBBl, AO, QS, QPT, ODC, 30 seconds at 94 ℃,30 seconds at 57 ℃ and 30 seconds at 72 ℃ and 24 cycles for detection of PMT, 1 minute at 94 ℃,30 seconds at 52 ℃ and 1 minute at 72 ℃.

A622 primer:

A622-07F 5'ATGGTTGTATCAGAGAAAAG

A622-05R 5'CCTTCTGCCTCTATCATCCTCCTG

NBBl primers:

NBBl-OlF 5'ATGTTTCCGCTC ATAATTCTG

NBBl-1365 5'TCTTCGCCCATGGCTTTTCGGTCT

AO primer:

AO ORT-1 5'CAAAACCAGATCGCTTGGTC

AO ORT–2 5'CACAGCACTTACACCACCTT

QS primers:

QSRT-1 5'CGGTGGAGCAAAAGTAAGTG

QSRT-2 5'GAAACGGAACAATCAAAGCA

QPT primer:

QPTRT-1 5'TCACTGCTACAGTGCATCCT

QPTRT-2 5'TTAGAGCTTTGCCGACACCT

ODC primer:

ODC RT-1 5'CGTCTCATTCCACATCGGTAGC

ODC RT-2 5'GGTGAGTAACAATGGCGGAAGT

PMT primers:

PMT RT-1 5'GCCATGATAATGGCAACGAG

PMT RT-2 5'TTAGCAGCGAGATAAGGGAA

as shown in fig. 6, a622 was not induced in a 622-silenced line. Other genes of known enzymes of the nicotine biosynthetic pathway are induced. These results provide evidence that a622 is classified in the nicotine alkaloid biosynthesis pathway and demonstrate that nicotine alkaloid content, particularly nicotine content, can be reduced by down-regulating a622 expression in a nicotine-producing plant cell.

Example 3: structure of inducible A622RNAi vector

Constitutive inhibition of a622 expression in hairy roots of tobacco significantly hindered root growth, hampering the analysis of alkaloids. To prevent this, an estradiol inducible gene expression system (XVE system) was developed. The XVE system produces RNAi hairpin molecules and inhibits the target gene only after the addition of an inducer (. beta. -estradiol) to the medium.

The RNAi region containing the A622 sense and antisense DNA fragments was excised from the pHANNIBAL-X plasmid using Xho I and Xba I and ligated into pBluescript KS which had been digested with Xho I and Xba I. The RNAi region was then excised with Xho I and Spe I and subcloned into the XVE vector pER8 between the Xhol and Spel sites within the MCS. (Zuo J. et al, planta J.,24: 265. sup. 273(2000)), a binary vector pXVE-A622RNAi was generated.

The T-DNA region of pXVE-A622RNAi (see FIG. 7) contains the chimeric estradiol-inducible expression cassette for the transcription factor XVE, the hpt selectable marker cassette, and the A622RNAi expression cassette activated by XVE under the control of the LexA-46 promoter.

Example 4 inhibition of A622 in tobacco hairy roots

The binary vector pXVE-A622RNAi was introduced into Agrobacterium rhizogenes strain 15834 by electroporation. The Agrobacterium rhizogenes was used to transform tobacco variety Petit Havana SRl plants using the leaf disc method as described by Kanegae et al, Plant Physiol.105(2):483-90 (1994). Transformed roots were selected for hygromycin resistance (15 mg/L in B5 medium). Transgenic hairy roots were grown in the dark at 27 ℃.

Transgenic hairy roots carrying T-DNA from pXVE-A622RNAi were grown in B5 medium for 10 days and cultured with 17-. beta. -estradiol (2. mu.M) for 4 days to induce gene silencing. RT-PCR analysis showed that A622 expression was effectively inhibited in tobacco hairy root lines A8 and A10 transformed with estradiol inducible suppression constructs. See fig. 8A.

Total RNA from hairy roots was extracted using a Plant RNeasy Mini kit (RNeasy Plant mini kit) (Qiagen). cDNA was synthesized for RT-PCR using random hexamers and Superscript first strand synthesis system (Invitrogen). RT-PCR was carried out using 2.5ng of cDNA as template and TaKaRa ExTaq enzyme (Takara Bio) for 22 cycles for detection of A622, 30 seconds at 94 ℃,30 seconds at 57 ℃ and 30 seconds at 72 ℃ and for 24 cycles for detection of alpha-tubulin, 1 minute at 94 ℃,30 seconds at 52 ℃ and 1 minute at 72 ℃.

Primers for a622 detection:

A622-07F 5'ATGGTTGTATC AGAGAAAAG

A622-05R 5'CCTTCTGCCTCTATCATCCTCCTG

primers for alpha-tubulin detection;

Tub RT-1 5'AGTTGGAGGAGGTGATGATG

Tub RT-2 5'TATGTGGGTCGCTCAATGTC

the hairy root line transformed with the inducible a622 suppression construct had no induced nicotine content (-) and no nicotine content (+) after estradiol-induced suppression. The RT-PCR graph in FIG. 8B shows that A622 expression has been partially inhibited prior to estradiol induction. This is particularly true in line # 8. The nicotine content varied between a622 inhibitory strains, but the nicotine content in a622 inhibitory strain was lower than that of wild type hairy roots.

Example 5: identification of NBBl as a NIC site regulated Gene

A cDNA chip prepared from a cDNA library derived from tobacco forest (Nicotiana sylvestris) (Katoh et al, Proc. Japan Acad. Vol.79, Ser.B5No.6, pp. 151-154(2003)) was used to search for a novel gene controlled by NIC sites regulated by nicotine biosynthesis.

N.sylvestris cDNAs were amplified by PCR and spotted onto mirrored slides (mirrored-coated slides) (7 star (type7star), Amersham) using an Amersham lucida spotter (Amersham lucida array spotter). Crosslinking by UV (120 mJ/m)²) The DNA was immobilized on the surface of the slide. Tobacco Burley 21 seedlings (WT and nicnic 2) were grown in an Agripot container (Kirin) on half strength B5 medium supplemented with 1.5% (W/V) sucrose and 0.35% (W/V) gellan gum (gellan gum) (Wako).

Eight weeks old young roots were harvested, immediately frozen in liquid nitrogen and stored at-80 ℃ until use. Total RNA was isolated from frozen roots using Plant RNeasy Mini kit (Plant RNeasy Mini kit) (Qiagen) and mRNA was purified using GenElute mRNA Miniprep kit (GenElute mRNA Miniprep kit) (Sigma). cDNA was synthesized using 0.4. mu.g of purified mRNA using Labelstar Array Kit (Labelstar Array Kit) in the presence of Cy3 or Cy5-dCTP (Amersham). Hybridization of the cDNA to microarray slides and washing after hybridization were performed using a Lucida Pro hybridization apparatus (Lucida Pro hybrid-machine) (Amersham). The chip was scanned using a FLA-8000scanner (FLA-8000scanner) (Fujifilm). Array images obtained by quantification of signal intensity using ArrayGauge software (ArrayGauge software) ((Fujifilm). FormicaPair-wise combinations in a positive-negative pairing, cDNA probes from wild-type and nic2 tobacco were labeled with Cy3 and Cy 5. normalization of hybridization signals based on colored total signal intensity. cDNA clones hybridizing to wild-type probes were identified significantly more than doubled compared to the nic2 probe and these included NBBl.

The full-length NBBl cDNA was obtained from tobacco total RNA by 5 '-and 3' -RACE using SMART RACE cDNA Amplification Kit (SMART RACE cDNA Amplification Kit) (Clontech).

Using ABI3100 Genetic Analyzer (Genetic Analyzer) (Applied Biosystems) andterminator v3.1cycle sequencing kit (Terminator v3.1cycle sequencing kit (Applied Biosystems) determined the nucleotide sequence of the NBBl cDNA insert on the duplex. The nucleotide sequence of NBBl is shown in SEQ ID NO 3. The amino acid sequence coded by the nucleotide sequence is shown as SEQ ID NO. 4. The protein sequence comprises a FAD binding motif. The putative vacuolar signal peptide (vacuolar signal peptide) is located at the N-terminus.

EXAMPLE 6 characterization of NBBl

Expression of NBBl in tobacco plants was detected by Northern blot analysis.

RNA was extracted from plant bodies that had been vapor-treated with methyl jasmonate, using tobacco variety Burley 21(Nicotiana tabacum cv. Burley 21) (hereinafter abbreviated as WT) and mutants nic, nic2 and nic2 that introduced mutations in the Burley 21 background. The cultivation is carried out in a sterile closed environment at 25 deg.C and 150 μmole photon/m²Under light, the plants were cultured on 1/2x B5 medium (3% sucrose, 0.3% gellan gum) for 2 months. To an Agripot container (80 m) containing plants³The solid medium capacity of (2) and 250m³Gas volume of (2) (Kirin Tokyo) 0.5mL of methyl jasmonate of 100. mu.M was added to complete the treatment of methyl jasmonate. The treatment times were 0h and 24 h. Root parts and leaf parts (2 nd to 6 th leaves on a plant body having 7 to 10 leaves) were collected from the plant body and immediately stored by freezing with liquid nitrogen.

RNA was extracted using RNeasy Mini kit (Qiagen) according to the manufacturer's protocol. Still, polyvinylpyrrolidine was added to the RLT to a concentration of 1%. Column operations were performed 2 times to increase RNA purity.

Northern blotting was performed according to the general method given by Sambrook and Russell (Sambrook, J. et al, Molecular Cloning, Cold spring harbor Laboratory, Chapter 7 (2001)).

A sequence fragment from 1278bp of the NBBl nucleotide sequence (SEQ ID NO:3) to its terminus (1759bp) was used as a probe template. Templates were prepared from cDNA clone amplifications by PCR using the following primers:

primer 1: GGAAAACTAACAACGGAATCTCT

Primer 2: GATCAAGCTATTGCTTTCCCT

Using the Bcabest labeling kit (Bcabest labeling kit) (Takara), the manufacturer's recommendations were followed³²P labels the probe. Hybridization was accomplished according to the manufacturer's protocol using ULTRAhyb (Ambion) as the buffer.

PMT probes were prepared from the PMT sequence of pcDNAII vector (Hibi et al, 1994) which had been cloned into E.coli. Plasmids were extracted and purified from E.coli using QIAprep Spin Miniprep Kit (QIAprep Spin Miniprep Kit) (Qiagen), treated with restriction enzymes Xbal and HindIII by a common method, run on an agarose gel, and an approximately 1.5kb DNA fragment was collected. The QIAquick Gel Extraction Kit (QIAquick Gel Extraction Kit) (Qiagen) was used for collection. Using the same method as for the NBBl probe³²P-tag the collected DNA fragments and hybridize. The results are shown in FIG. 9.

FIG. 9 clearly shows that NBBl and PMT are expressed in the same way in tobacco plants. NBB1 is involved in nicotine biosynthesis as evidenced by NBBl being under NIC gene regulation like PMT and a622, and it shows similar expression pattern as PMT and a 622.

EXAMPLE 7 phylogenetic analysis of NBBl

The NBBl polypeptide has 25% identity and 60% homology to the Eschschschscholzia californica berberinebridge enzyme (BBE). (Dittrich H. et al, Proc. Natl. Acad. ScI USA, Vol.889969-9973 (1991)). The NBBl polypeptide to EcBBE alignment is shown in figure 10.

Phylogenetic trees were constructed using NBB1 polypeptide sequences and Plant BBE-like polypeptide sequences (based on Carter and Thornburg, Plant Physiol.134,460-469 (2004)). Systematic analysis was performed using the CLUSTAL W program using the neighbor joining method. The numbers show bootstrap values for 1,000 replicates. The sequence used was EcBBE, California poppy BBE (GenBank accession No. AF 005655); PsBBE, poppy (Papaversomniferum) possible reticuline oxidase AF 025430; BsBBE, barberry (berberissolonifera) BBE (AF 049347); VuCPRD2, cowpea (Vigna unguiculata) drought-induced protein (AB 056448); NspNEC5, Nicotiana species Nectarin V (AF503441/AF 503442); HaCHOX, sunflower (Helianthus annuus) carbohydrate oxidase (AF 472609); LsCHOX, lettuce (Lactuca sativa) glycosyloxidase (AF 472608); and 27 Arabidopsis genes (Atlg01980, Atlgl 770, 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 At5g 44440).

The results are shown in FIG. 11. The three known BBEs form independent clades and are emphasized and pointed out as "true BBEs. The NBBl sequence does not have a high degree of similarity to any BBE or BBE-like protein (BBE-like protein) and is separated from other sequences at the base of the tree. The only other described BBE-like proteins are from nicotiana, nectarinV, the described proteins are present in nectar of the cross-decorated (hybrid organization) nicotiana cinnamomea (langsdorffii xn. sanderare), Carter and Thornburg (2004), grouped with cowpea drought-inducing proteins and several recognized BBE-like proteins from arabidopsis thaliana. Since the nectar of the decorated tobacco lacks alkaloids and nectarine V has glucose oxidase activity, it can be concluded that nectarine V is involved in antibacterial defenses in flowers and may not be involved in alkaloid synthesis. And Id.

EXAMPLE 8 preparation of NBBl inhibitory constructs

The 342-bp DNA fragment of NBBl cDNA was PCR amplified with the following primers and cloned into pGEM-T vector.

Antisense strand

NBBI-20F-EcoRI 5'CCGGAATTCGCACAGTGGAATGAAGAGGACG 3'

NBBl-18R-XhoI 5'CCGCTCGAGGCGTTGAACCAAGCATAGGAGG 3'

Sense strand

NBB1-16F-ClaI 5'CCATCGATGCACAGTGGAATGAAGAGGACG 3'

NBB-19R-bal 5'GCTCTAGAGCGTTGAACCAAGCATAGGAGG 3'

The final PCR product was digested with EcoRI and Xhol for antisense insertion and CIaI and xbal for sense strand insertion. The sense DNA fragment was subcloned into pHANNIBAL-X, followed by insertion of the antisense fragment. The resulting plasmid contains the inverted repeat DNA sequence of the NBBl fragment separated by the Pdk intron.

The RNAi region was excised from pHANNIBAL-X with BamH I and Sac I and ligated into pBI121 to replace the GUS coding region, resulting in the binary vector pRNAi-NBBl. The T-DNA region of pHANNIBAL-NBBl 3 '(see FIG. 12) contains the nptII selectable marker cassette and the hairpin RNAi expression cassette with a double stranded region comparable to the NBBl 3' fragment.

Example 9: NBBl inhibition in tobacco BY-2 cells

Methyl jasmonate treatment of tobacco BY-2 cells induced the expression of enzyme genes known in the nicotine biosynthesis pathway in addition to NBBl. The effect of NBBl inhibition was examined using BY-2 cells.

Vector pRNAi-NBB1 was introduced into Agrobacterium tumefaciens strain EHAl05, which was used to transform tobacco BY-2 cells. BY-2 cells were cultured in 100ml of modified LS medium. Mu.l of Agrobacterium tumefaciens cells in YEB medium were added to 4ml of BY-2 cells and cultured in the dark at 27 ℃ for 40 hours. Infected tobacco cells were washed twice with modified LS medium and the washed tobacco cells were spread on modified LS agar medium containing kanamycin (50mg/1) and carbenicillin (250 mg/1). After 2 weeks of dark culture at 27 ℃, the growing tobacco calli were transferred to fresh LS selection medium with the same antibiotics and dark cultured for another week at 27 ℃. The growing tobacco cells were transferred to modified LS medium without antibiotics. The transformed tobacco cells were sub-cultured at 7-day intervals.

The cultured tobacco cells were cultured in modified LS medium without 2,4-D at 27 ℃ for 12 hours. To 100mL of the tobacco suspension culture, 100. mu.l of methyl jasmonate dissolved in DMSO was added to a final concentration of 50. mu.M, and the tobacco cells were cultured for another 48 hours. MeJA treated cells were filtered, collected, and lyophilized. To 100mg of the freeze-dried sample was added 3ml of 0.1N sulfuric acid. The mixture was sonicated for 15 minutes and filtered. To 1ml of the filtrate was added a 28% ammonium (ammonium) solution and centrifuged at 15000rpm for 10 minutes. To an Extrelut-1 column (Merck) 1ml of supernatant was added and eluted with 6ml of chloroform. The eluate was dried under reduced pressure at 37 ℃ using an evaporator (Taitec Concentrator TC-8). The dried sample was dissolved in 50. mu.l of 0.1% dodecane in ethanol. The samples were analyzed by gas chromatography equipped with a capillary column (GC-14B) and FID detector. A RESTEC Rtx-5Amine column (Restec) was used as the capillary column. The column temperature was maintained at 100 ℃ for 10 minutes, increased to 150 ℃ at a rate of 25 ℃/minute, maintained at 150 ℃ for 1 minute, increased to 170 ℃ at a rate of 1 ℃/minute, maintained at 170 ℃ for 2 minutes, increased to 300 ℃ at a rate of 30 ℃/minute, and then maintained at 300 ℃ for 10 minutes. Both the injection and detection temperatures were 300 ℃. Mu.l of each sample was injected and nicotine alkaloid was quantified by the internal standard method.

Compared with wild type tobacco cells, NBBl-inhibited BY-2 cell lines (N37 and N40) showed a greatly reduced accumulation of nicotine alkaloids after induction with methyl jasmonate (see FIG. 5).

To determine whether the reduction in alkaloid accumulation in NBB1-RNAi lines was specifically associated with a reduction in NBBl expression, rather than indirectly affecting expression of known enzyme genes in the nicotine biosynthetic pathway, the expression levels of NBBl and other genes were measured in both methyl jasmonate treated lines, transgenic lines, and control lines.

Total RNAs were isolated from wild type and transgenic BY-2 cell lines that had been treated with 50uM MeJA for 48 h. The RNA level of a specific gene was determined by RT-PCR. The results are shown in FIG. 6.

No induction of NBBl was observed in NBBl silencing lines, but induction of known genes of the nicotine biosynthesis pathway still occurred, such as induction of a 622. It was also noted that NBBl was not affected in the a662 suppression line.

These results demonstrate that NBBl reductase is involved in the nicotine alkaloid biosynthesis pathway and that nicotine alkaloid content, particularly nicotine content, can be reduced by down-regulating NBBl expression in a nicotine-producing plant cell.

Example 10 NBBl inhibition in tobacco hairy roots

Tobacco SR-1 hairy roots accumulate nicotine as the main alkaloid. The effect of NBBl inhibition on alkaloid accumulation in hairy roots was investigated.

The binary vector pRNAi-NBB 13' was introduced into Agrobacterium rhizogenes strain 15834 by electroporation. Transformation of tobacco variant Petit Havana SRl plants with Agrobacterium rhizogenes by the leaf disc method, inhibition of A622 in tobacco hairy roots as described above. Hairy roots were selected and cultured as described above and alkaloids were extracted, purified and analyzed.

When NBBl expression was inhibited by RNAi, transgenic roots (HN6, HNl9, HN20 and HN29) contained very low nicotine levels and reduced anatabine levels compared to control cell lines (see figure 13).

The transgenic hairy roots carrying pHANNIBAL-NBBl 3' were cultured in B5 medium for two weeks and analyzed for gene expression by RT-PCR. NBBl expression was specifically inhibited in four transgenic lines (see fig. 14). NBBl expression of other enzyme genes of the nicotine biosynthetic pathway was not affected.

The results demonstrate that the reduction in nicotine accumulation results from a reduction in NBBl expression, not from expression of known enzyme genes lacking the nicotine biosynthetic pathway.

Example 11 NBBl inhibition in transgenic tobacco plants

Two attB-NBBl fragments were amplified by PCR from NBBl cDNA on pGEM-T vector using a set of primers NBB1-aatBl and attBl linker, and NBBl-attB2 and attB2 linker.

Gene specific primer

NBBl-attB1 5'AAAAAGCAGGCTTCGAAGGAGATAGAACCATGGTTCCGCTCATAATTCTGATCAGCTT

NBBl-attB2 5'AGAAAGCTGGGTCTTCACTGCTATACTTGTGCTCTTGA

Linker primer

attBl linker 5' GGGGACAAGTTTGTACAAAAAAGC AGGCT

attB2 linker 5' GGGGACCACTTTGTACAAGAAAGCTGGGT

PCR conditions as recommended by the manufacturer were used. The expression clone pDONR221-NBBl-1 was generated by BP recombination reaction of attB-NBBl PCR product and pDONR221 (Invitrogen).

The NBBl Open Reading Frame (ORF) was transferred from the pDONR221-NBBl-l vector to the digested GATEWAY binary vector pANDA 35HK, expressing dsRNA together with the GUS partial fragment under the CaMV35S promoter by LR reaction (Dr. KoShimamoto, NAIST). The resulting NBBl RNAi vector is called pANDA-NBBl full length.

The full-length T-DNA of pANDA-NBB1 (see FIG. 15) contained the nptll selectable marker cassette, the NBBl RNAi cassette, in which the full-length coding region of NBBl was present as inverted repeats and separated by the GUS linker, and the hpt selectable marker cassette.

The binary vector pANDA-NBBl was introduced into Agrobacterium tumefaciens strain EHAl05 in its full length by electroporation. Transformation of tobacco variety Petit Havana SRl plants with Agrobacterium tumefaciens by the leaf disc method was essentially as described by Kanegae et al, plantaphysiol.105, 483-490 (1994). Hygromycin resistance (30 mg/L in MS medium) was used for selection. Transgenic plants were regenerated from the leaf disks and grown in a growth chamber at 27 ℃ under continuous light.

Leaf tissue from 36-day-old T0 plants was collected. The alkaloids were extracted, purified and analyzed as described above. The level of nicotine was reduced in leaves of plants from lines that had been fully transformed with NBBl inhibiting pANDA-NBBl compared to wild type. The leaves of transgenic lines (#6, #14 and #22) contained nicotine levels of only about 16% of the levels of wild-type plant leaves.

Claims (21)

1. A method of reducing an alkaloid in a plant, comprising down-regulating expression of a nicotine biosynthetic pathway gene relative to a non-transformed control plant, wherein the nicotine biosynthetic pathway gene is an a622 gene, the a622 gene comprising a nucleotide sequence selected from the group consisting of:

(a) 1, SEQ ID NO;

(b) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO. 2; and

(c) a nucleotide sequence encoding a polypeptide having one or more conservative amino acid substitutions as compared to the polypeptide having the amino acid sequence set forth in SEQ ID NO. 2 and having A622 expression.

2. The method of claim 1, wherein a622 is down-regulated by introducing into a plant cell of the plant a nucleic acid that expresses a short interfering RNA that inhibits the expression of a 622.

3. The method of claim 1, wherein a622 is down-regulated by introducing into a plant cell of the plant an enzymatic RNA molecule that cleaves a622mRNA transcript.

4. The method of claim 1, wherein a622 is down-regulated by introducing into a plant cell of the plant a nucleic acid construct comprising in the 5 'to 3' direction a promoter operably linked to a heterologous nucleic acid encoding a622 and a terminator.

5. A process according to any one of claims 1 to 4, wherein the alkaloid is nicotine.

6. A process according to any one of claims 1 to 4, wherein the alkaloid is anatabine.

7. The method according to any one of claims 1 to 6, wherein the plant is a tobacco plant.

8. A genetically engineered plant cell having reduced a622 expression, wherein a622 is encoded by a nucleotide sequence selected from the group consisting of:

(a) 1, SEQ ID NO;

(b) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO. 2; and

(c) a nucleotide sequence encoding a polypeptide having one or more conservative amino acid substitutions as compared to the polypeptide having the amino acid sequence set forth in SEQ ID NO. 2 and having A622 expression.

9. The plant cell of claim 8, wherein the plant cell is isolated from a genetically engineered tobacco plant.

10. The plant cell of claim 8 or 9, wherein the alkaloid is nicotine.

11. The plant cell of claim 8 or 9, wherein said alkaloid is anatabine.

12. An alkaloid reduced product produced from a plant comprising the plant cell of any one of claims 8-11, wherein the product is selected from the group consisting of: tobacco products, food ingredients, feed products, feed ingredients, nutritional supplements and biofuels.

13. The reduced alkaloid product of claim 12, wherein said product is selected from the group consisting of: cigarettes, cigarette-used tobacco leaves, cigars, cigar-used tobacco leaves, pipe tobacco, chewing tobacco, snuff, whole tobacco leaves, cut tobacco and shredded tobacco.

14. A method of reducing alkaloid content in a plant comprising down-regulating a nicotine biosynthesis gene and a622, wherein a622 is encoded by a nucleotide sequence selected from the group consisting of:

(a) 1, SEQ ID NO;

(b) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO. 2; and

(c) a nucleotide sequence encoding a polypeptide having one or more conservative amino acid substitutions as compared to the polypeptide having the amino acid sequence set forth in SEQ ID NO. 2 and having A622 expression.

15. The method of claim 14, wherein the nicotine biosynthesis gene is selected from the group consisting of: aspartate oxidase, quinolinate synthase, quinolinate phosphoribosyltransferase, ornithine decarboxylase, putrescine N-methyltransferase and diamine oxidase.

16. A genetically engineered plant cell isolated from a plant having reduced a622 expression and reduced alkaloid content, wherein a622 is encoded by a nucleotide sequence selected from the group consisting of:

(a) 1, SEQ ID NO;

(b) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO. 2; and

(c) a nucleotide sequence encoding a polypeptide having one or more conservative amino acid substitutions as compared to the polypeptide having the amino acid sequence shown in SEQ ID NO. 2 and having A622 expression.

17. The plant cell of claim 16, wherein the plant cell is isolated from a genetically engineered tobacco plant.

18. The plant cell of claim 16 or 17, wherein said alkaloid is nicotine.

19. The plant cell of claim 16 or 17, wherein said alkaloid is anatabine.

20. An alkaloid reduced product produced from a plant comprising the plant cell of any one of claims 16-19, wherein the product is selected from the group consisting of: tobacco products, food ingredients, feed products, feed ingredients, nutritional supplements and biofuels.

21. The reduced alkaloid product of claim 20, wherein said product is selected from the group consisting of: cigarettes, cigarette-used tobacco leaves, cigars, cigar-used tobacco leaves, pipe tobacco, chewing tobacco, snuff, whole tobacco leaves, cut tobacco and shredded tobacco.