HK1154629B - Tobacco nicotine demethylase genomic clone and uses thereof - Google Patents

Description

Tobacco nicotine demethylase genomic clone and application thereof

The invention relates to a division application of Chinese patent application 200580019669.4, namely the genomic clone of tobacco nicotine demethylase and the application thereof, wherein the international application date is 4/20/2005.

The present invention relates to nucleic acid sequences encoding nicotine demethylase enzymes and methods of using those nucleic acid sequences to alter plant phenotypes.

Background

Cytochrome p450s catalyzes an enzymatic reaction for a wide range of chemically dissimilar substrates, including oxidative, peroxidative and reductive metabolism of endogenous and xenobiotic substrates. In plants, p450s is involved in biochemical pathways including the synthesis of plant products such as phenyl propanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides and glucosinolates (Chappell, Annu. Rev. plant Physiol. plant mol. biol. 46: 521-547, 1995). Cytochrome p450s, also known as p450 heme-thiolate protein, generally acts as a terminal oxidase in the multicomponent electron transport chain known as the p 450-containing monooxygenase system. Specific reactions catalyzed by these enzyme systems include demethylation, hydroxylation, epoxidation, N-oxidation, thiooxidation, N-, S-, and O-dealkylation, desulfurization, deamination, and reduction of azo, nitro, and N-oxide groups.

The diverse role of the Nicotiana (Nicotiana) plant p450 enzymes is involved in the effects on a variety of plant metabolites such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, glucosinolates and many other chemical entities. Some p450 enzymes can affect the composition of plant metabolites. For example, it has long been desirable to modify the pattern of selected fatty acids of plants by breeding to improve the flavor and aroma of certain plants; however, little is known about the mechanisms involved in controlling the levels of these leaf components. The down-regulation or up-regulation of p450 enzymes associated with fatty acid changes may promote the accumulation of desirable fatty acids, which provides a more preferred quality of the leaf phenotype.

The function of p450 enzymes and their further role in plant components is still under discovery. For example, a particular class of p450 enzymes has been found to catalyze the breakdown of fatty acids into volatile C6-and C9-aldehydes and β -alcohols, which are the major contributing factors to the "greenish" odor of fruits and vegetables. The levels of other novel target p450s can be altered to improve the quality of leaf components by altering the lipid composition and associated catabolites in nicotiana leaves. Some of these leaf components are affected by senescence which stimulates leaf quality maturation. Still other reports have shown that the p450s enzyme has a functional role in altering fatty acids involved in plant-pathogen interactions and disease resistance.

In other cases, p450 enzymes have been shown to be involved in alkaloid biosynthesis. Nornicotine, a minor alkaloid found in tobacco (Nicotiana tabacum), is produced by p 450-mediated demethylation of nicotine followed by acylation and nitrosation at the N-site, thereby producing a series of N-acyl and N-nitrosonornicotine as provided by applicants' patent application, which claims priority thereto and is incorporated herein by reference. The N-demethylation catalyzed by p450 demethylase is believed to be a major source in nornicotine biosynthesis in nicotiana.

There is a need in the art for agents and methods for improving plant phenotype. In particular, there is a need for agents and methods that improve nicotine demethylase. The present invention provides a number of strategies for improving the expression of nicotine demethylase.

Summary of The Invention

The applicant has identified and characterized genomic clones of nicotine demethylase from tobacco. Included herein are the sequence of the coding region of the nicotine demethylase protein, the 3 'untranslated sequence ("3' UTR"), the single intron, and the nicotine demethylase gene promoter and its transcriptional regulatory sequences (figure 1). Also described is the use of these sequences to produce transgenic plants having altered levels of nornicotine or N' -nitrosonornicotine ("NNN"), or both, relative to control plants.

Thus, in a first aspect, the invention features an isolated nucleic acid molecule, e.g., a DNA sequence comprising a nucleotide sequence encoding a nicotine demethylase. In desirable embodiments, the nucleotide sequence of the first aspect is substantially identical to a nucleotide sequence encoding a tobacco nicotine demethylase, such as a tobacco nicotine demethylase comprising a nucleotide sequence identical to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO: 3, or a nucleotide sequence comprising at least 70% identical to the nucleotide sequence of SEQ ID NO:1, or nucleotide 2010-2949 and/or 3947-4562 comprising SEQ ID NO:1 or SEQ ID NO: 3. The isolated nucleic acid molecule of the first aspect of the invention is, for example, operably linked to a promoter functional in a plant cell and desirably contained in an expression vector. In other desirable embodiments, the expression vector is contained in a cell, such as a plant cell. Ideally, plant cells, such as tobacco plant cells, are included in the plant. In another desirable embodiment, the invention features a seed from a plant comprising an expression vector, such as a tobacco seed, wherein the seed includes an isolated nucleic acid molecule that hybridizes under stringent conditions to the mature polypeptide of SEQ ID NO: 3, operably linked to a heterologous promoter sequence. Furthermore, the invention features plants from germinated seeds containing the expression vectors, green or cured (cured) leaves from the plants, and articles made from the leaves.

In another desirable embodiment, the nucleotide sequence comprises a sequence that hybridizes under stringent conditions to the complement of SEQ ID NO:1 and/or SEQ ID NO: 3, or the complement of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO: 3. Desirably, the nucleotide sequence encodes a nicotine demethylase that differs from the nucleotide sequence set forth in SEQ ID NO: 2 are substantially identical in amino acid sequence. In another desirable embodiment of the first aspect of the invention, the nicotine demethylase has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 2 or a fragment of a nicotine demethylase having altered (e.g., reduced) enzymatic activity as compared to the full-length polypeptide, has at least 70% identity. Desirably, the nicotine demethylase comprises SEQ ID NO: 2.

In another aspect, the invention features an isolated nucleic acid molecule comprising a promoter that hybridizes under stringent conditions to the complement of SEQ ID NO: 6. Ideally, the promoter is (i) induced after treatment with ethylene or during senescence; and (ii) comprises (a) SEQ ID NO:1, or (b) at least 200 consecutive base pairs that hybridize to a polypeptide consisting of SEQ ID NO:1, or (c) a 20 base pair nucleotide moiety that is identical in sequence to the 200 consecutive base pairs defined in base pairs 1-2009 of SEQ ID NO: the 20 contiguous base pair portions proposed in base pairs 1-2009 of 1 are identical in sequence.

Another aspect of the invention features an isolated nucleic acid promoter that includes a sequence identical to SEQ ID NO: 6 has a nucleotide sequence of 50% or more sequence identity. Ideally, the isolated nucleic acid promoter is induced after treatment with ethylene, or during senescence, and comprises, for example, the amino acid sequence of SEQ ID NO: 6. Alternatively, the promoter may comprise a sequence derivable from SEQ ID NO: 6, wherein the fragment drives transcription of a heterologous gene or reduces or alters nicotine demethylase activity (e.g., silences gene expression). In desirable embodiments, the promoter sequence is operably linked to a heterologous nucleic acid sequence, and may, for example, be comprised in an expression vector. In other desirable embodiments, the expression vector is contained in a cell, such as a plant cell. Ideally, plant cells, such as tobacco plant cells, are included in the plant. In another desirable embodiment, the invention features a seed from a plant comprising an expression vector, such as a tobacco seed, wherein the seed comprises an isolated nucleic acid molecule that hybridizes, under stringent conditions, to the full complement of SEQ ID NO: 6, operably linked to a heterologous nucleic acid sequence. Furthermore, the invention features a plant from a germinated seed comprising a promoter of this aspect of the invention, a green or processed leaf from said plant, or an article made from said leaf.

Another aspect of the invention features a method of expressing a heterologous gene in a plant. The method comprises (i) introducing into a plant cell a vector comprising a promoter sequence that hybridizes to SEQ ID NO: 6, which is operably linked to a heterologous nucleic acid sequence, has 50% or more sequence identity; and (ii) regenerating a plant from the cell. In addition, the method may comprise the step of sexually transmitting the vector to progeny, and further may comprise the step of collecting seeds produced from the progeny.

In another aspect, the invention features a method of reducing expression of nicotine demethylase in a tobacco plant. The method comprises the following steps: (i) combining a nucleic acid sequence comprising SEQ ID NO: 6 or a sequence derivable from SEQ ID NO: 6 into a tobacco plant, and (ii) expressing the vector in the tobacco plant. In a desirable embodiment of the method, expression of tobacco demethylase is silenced. In other desirable embodiments, the vector expresses RNA, such as antisense RNA or an RNA molecule capable of inducing RNA interference (RNAi).

In another desirable aspect, the invention features an isolated nucleic acid molecule comprising an intron that hybridizes under stringent conditions to the complement of SEQ ID NO: 5 or a fragment thereof that reduces or alters the enzymatic activity (e.g., silences gene expression) of a nicotine demethylase or can serve as a molecular marker to identify a nicotine demethylase nucleic acid sequence. In desirable embodiments, the intron comprises (a) the amino acid sequence of SEQ ID NO:1, or (b) at least 200 consecutive base pairs that hybridize to SEQ ID NO:1 base pair 2950-: the 20 consecutive base pair portions proposed in base pairs 2950-3946 of 1 are identical in sequence.

Another desirable aspect of the invention features an isolated nucleic acid intron that includes a sequence identical to seq id NO: 5 or a fragment thereof, that reduces or alters the enzymatic activity of a nicotine demethylase (e.g., silenced gene expression) or can serve as a molecular marker to identify a nicotine demethylase nucleic acid sequence. Silencing gene expression may, for example, include homologous recombination or mutation resulting in a gene product that does not have nicotine demethylase activity. In particular, the intron can include SEQ ID NO: 5 or a sequence which can be derived from SEQ ID NO: 5 the fragment obtained. Ideally, the isolated nucleic acid molecule comprising the intron is operably linked to a heterologous nucleic acid sequence and the sequence is desirably contained in an expression vector. In another embodiment, the expression vector is comprised in a cell, such as a plant cell. In particular, the cell may be a tobacco cell. A plant, such as a tobacco plant, comprising a plant cell comprising SEQ ID NO: 5 or a sequence derivable from SEQ ID NO: 5 the fragment obtained. In addition, seeds from plants, such as tobacco seeds, comprising introns that hybridize under stringent conditions to the amino acid sequence of SEQ id no: 5 operably linked to a heterologous nucleic acid sequence. Furthermore, the invention features plants from germinated seeds comprising the introns of this aspect of the invention, green or processed leaves from said plants, and articles made from green or processed leaves.

Another aspect of the invention features a method of expressing an intron in a plant. The method comprises (i) introducing into a plant cell an expression vector comprising the nucleotide sequence of SEQ ID NO: 5 or a sequence derivable from SEQ ID NO: 5 the fragment obtained; and (ii) regenerating a plant from the cell. In desirable embodiments, the method further comprises (iii) delivering the vector to progeny by sexual means, and may include the additional step of collecting seed produced by the progeny. The methods desirably include, for example, regenerating plants from germinated seeds, green or processed leaves from the plants, and methods of preparing articles from the leaves.

In another aspect, the invention features a method of reducing expression of nicotine demethylase in a tobacco plant. The method comprises the steps of (i) introducing into a tobacco plant a vector comprising the nucleotide sequence of SEQ ID NO: 5 or a sequence derivable from SEQ ID NO: 5, and (ii) expressing the vector in a tobacco plant. In a desirable embodiment of the method, expression of nicotine demethylase is silenced. In other desirable embodiments, the vector expresses RNA, such as antisense RNA or an RNA molecule capable of inducing RNA interference (RNAi).

In another aspect, the invention features an isolated nucleic acid molecule comprising an untranslated region that hybridizes under stringent conditions to the complement of SEQ ID NO: 7 or a fragment thereof, said SEQ ID NO: 7 or fragments thereof can alter the expression pattern of the gene, reduce or alter nicotine demethylase enzyme activity (e.g., silence gene expression), or can be used as a marker to identify nicotine demethylase nucleic acid sequences. In a desirable embodiment of this aspect of the invention, the untranslated region includes (a) the amino acid sequence of SEQ ID NO:1, or (b) at least 200 consecutive base pairs that hybridize to the nucleotide sequence of SEQ ID NO:1 base pair 4563-: 1 base pairs 4563-6347 proposes sequences in which 20 consecutive base pairs are identical.

Another desirable aspect of the invention features an isolated nucleic acid untranslated region comprising a nucleotide sequence that hybridizes to SEQ ID NO: 7 has a sequence identity of 50% or more. Desirably, the untranslated region comprises SEQ ID NO: 7 or said untranslated region comprises a sequence derivable from SEQ ID NO: 7, which can alter the expression pattern of the gene, reduce or alter the enzymatic activity of the nicotine demethylase (e.g., silence gene expression), or can be used as a marker to identify a nicotine demethylase nucleic acid sequence. The untranslated region is desirably operably linked to a heterologous nucleic acid sequence and may be contained in an expression vector. Furthermore, the expression vector is desirably comprised in a cell, such as a plant cell, e.g., a tobacco cell. Another desirable embodiment of the invention features a plant, such as a tobacco plant, that includes a plant cell that includes a vector that includes an isolated nucleic acid sequence that hybridizes to SEQ id no: 7 has 50% or more sequence identity and is operably linked to a heterologous nucleic acid sequence.

The invention also features a seed from a plant, such as a tobacco seed, wherein the seed includes an untranslated region that hybridizes under stringent conditions to the amino acid sequence of SEQ ID NO: 7 operably linked to a heterologous nucleic acid sequence. Furthermore, the invention features plants from germinated seeds comprising the untranslated regions of this aspect of the invention, green or processed leaves from said plants, and articles made from green or processed leaves.

In another aspect, the invention features a method of expressing an untranslated region in a plant. The method comprises (i) introducing into a plant cell a vector comprising an isolated nucleic acid sequence that hybridizes to SEQ ID NO: 7 has 50% or more sequence identity and is operably linked to a heterologous nucleic acid sequence; and (ii) regenerating a plant from the cell. In addition, the method may further comprise (iii) delivering the vector to progeny by sexual means, and desirably, comprising the further step of collecting seeds produced by the progeny. The method desirably includes regenerating a plant from the germinated seed, green or processed leaves from the plant, and a method of preparing an article from the green or processed leaves.

In addition, the invention features methods of reducing the expression of or altering the enzymatic activity of a nicotine demethylase in a tobacco plant. The method comprises the following steps: (i) introducing a vector into a tobacco plant, the vector comprising an isolated nucleic acid sequence that hybridizes to SEQ ID NO: 7 and is operably linked to a heterologous nucleic acid sequence and (ii) expressing the vector in a tobacco plant. Ideally, expression of nicotine demethylase is silenced. In other desirable embodiments, the vector expresses RNA, such as antisense RNA or an RNA molecule capable of inducing RNA interference (RNAi).

Another aspect of the invention features an expression vector that includes a nucleic acid molecule that includes a nucleotide sequence encoding a nicotine demethylase, wherein the vector is capable of directing the expression of the nicotine demethylase encoded by the isolated nucleic acid molecule. Desirably, the vector comprises SEQ ID NO:1 or SEQ ID NO: 3. In other desirable embodiments, the invention features a plant or plant component, such as a tobacco plant or plant component (e.g., tobacco leaf or stem), that includes a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that demethylates nicotine.

Another aspect of the invention features a cell comprising an isolated nucleic acid molecule that includes a nucleotide sequence encoding a nicotine demethylase. Ideally, the cell is a plant cell or a bacterial cell, such as an Agrobacterium.

Another aspect of the invention features a plant or plant component (e.g., tobacco leaf or stem) that includes an isolated nucleic acid molecule encoding a nicotine demethylase, wherein the nucleic acid molecule is expressed in the plant or plant component. Desirably, the plant or plant component is an angiosperm, a dicot, a solanaceous plant, or a nicotiana species. Other desirable embodiments of this aspect are seeds or cells from the plant or plant component, as well as green or processed leaves from the plant and articles prepared therefrom.

In a further aspect, the invention features a tobacco plant having reduced expression of a nucleic acid sequence encoding a polypeptide, such as a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and demethylating nicotine, wherein the reduced expression (or reduced enzymatic activity) reduces the level of nornicotine in a plant. In desirable embodiments, the tobacco plant is a transgenic plant, such as a plant comprising a transgene that, when expressed in a transgenic plant, silences gene expression of an endogenous tobacco nicotine demethylase.

In particular, the transgenic plant desirably includes one or more of: a transgene that expresses an antisense molecule of a tobacco nicotine demethylase or an RNA molecule capable of inducing RNA interference (RNAi); a transgene that, when expressed in a transgenic plant, co-inhibits expression of tobacco nicotine demethylase; encodes a dominant negative (dominant negative) negative gene product, such as SEQ ID NO: 2 in a mutated form; in the case of a polypeptide encoding SEQ ID NO: 2; a deletion in a gene encoding tobacco nicotine demethylase; and an insertion in a gene encoding a tobacco nicotine demethylase.

In other desirable embodiments, the reduced expression of the nucleic acid sequence encoding the polypeptide occurs at the transcriptional level, at the translational level, or at the post-translational level.

Another aspect of the invention features a tobacco plant comprising a recombinant expression cassette stably integrated into its genome, wherein the expression cassette is capable of effecting a reduction in nicotine demethylase activity. The seeds of the tobacco plant are a feature in desirable embodiments. Other desirable embodiments include green or processed leaves from the plant and articles made therefrom.

Another aspect of the invention features a method of expressing a tobacco nicotine demethylase in a plant. The method comprises (i) introducing into a plant cell an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a nicotine demethylase; and (ii) regenerating a plant from said cell. In desirable embodiments, the method is characterized by the delivery of the vector to progeny by sexual means, and desirably further comprises the additional step of collecting seed produced by the progeny. Additional desirable embodiments include plants from germinated seeds, green or processed leaves from the plants, or articles prepared from the green or processed leaves.

Another aspect of the invention features a substantially pure tobacco nicotine demethylase. Desirably, the tobacco nicotine demethylase comprises an amino acid sequence substantially identical to SEQ ID NO: 2 or an amino acid sequence comprising at least 70% identity to the amino acid sequence of seq id NO: 2. In desirable embodiments, the tobacco nicotine demethylase, upon expression in a plant cell, converts nicotine to nornicotine. In other desirable embodiments, the tobacco nicotine demethylase, after expression in a plant cell, is predominantly localized in the leaf, or the tobacco nicotine demethylase is induced by ethylene or expressed during plant senescence.

In another aspect, the invention features a substantially pure antibody that specifically recognizes and binds tobacco nicotine demethylase. Desirably, the antibody recognizes and binds to a recombinant tobacco nicotine demethylase, e.g., comprising the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.

Another aspect of the invention features a method of producing a tobacco nicotine demethylase. The method comprises the following steps: (a) providing a cell transformed with an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that demethylates nicotine; (b) culturing the transformed cell under conditions in which the isolated nucleic acid molecule is expressed; and (c) recovering the tobacco nicotine demethylase. The invention also features a recombinant tobacco nicotine demethylase produced according to this method.

In another aspect, the invention features a method of isolating a tobacco nicotine demethylase, or fragment thereof. The method comprises the following steps: (a) bringing SEQ ID NOS: 1, 3, 5, 6, or 7 or a portion thereof with a nucleic acid preparation from a plant cell under hybridization conditions that provide for detection of a nucleic acid sequence that hybridizes to a nucleic acid sequence as set forth in SEQ ID NOS: 1, 3, 5, 6, or 7 have at least 70% or greater sequence identity; and (b) isolating the hybridized nucleic acid sequence.

In another aspect, the invention features another method of isolating a tobacco nicotine demethylase, or fragment thereof. The method comprises the following steps: (a) providing a sample of plant cell DNA; (b) providing a pair of oligonucleotides that hybridizes to a nucleic acid having the sequence of SEQ id no:1, 3, 5, 6, or 7 has sequence identity over a region of the nucleic acid molecule; (c) contacting the oligonucleotide pair with plant cell DNA under conditions suitable for polymerase chain reaction mediated DNA amplification; and (d) isolating the amplified tobacco nicotine demethylase or fragment thereof. In a desirable embodiment of this aspect, a sample of cDNA prepared from a plant cell is subjected to an amplification step. In another desirable embodiment, the tobacco nicotine demethylase encodes a polypeptide that hybridizes to SEQ ID NO: 2 has at least 70% identity.

Another aspect of the invention features a method of reducing expression of a tobacco nicotine demethylase in a plant or plant component. The method comprises the following steps: (a) introducing a transgene encoding a tobacco nicotine demethylase into a plant cell to produce a transformed plant cell, said transgene being operably linked to a promoter functional in the plant cell; and (b) regenerating a plant or plant component from the transformed plant cell, wherein the tobacco nicotine demethylase is expressed in the cell of the plant or plant component, thereby reducing expression of the tobacco nicotine demethylase in the plant or plant component. In a particular embodiment of this aspect of the invention, the transgene encoding tobacco nicotine demethylase is expressed constitutively or inducibly, e.g., in a tissue-specific, cell-specific or organ-specific manner. In another embodiment of this aspect of the invention, expression of the transgene co-inhibits expression of an endogenous tobacco nicotine demethylase.

Another aspect of the invention features another method of reducing expression of a tobacco nicotine demethylase in a plant or plant component. The method comprises the following steps: (a) introducing into a plant cell a transgene encoding an antisense coding sequence of a tobacco nicotine demethylase or an RNA molecule capable of inducing RNA interference (RNAi) operably linked to a promoter functional in the plant cell to produce a transformed plant cell; and (b) regenerating a plant or plant component from the transformed plant cell, wherein an antisense coding sequence to a coding sequence for a tobacco nicotine demethylase or an RNA molecule capable of inducing RNA interference (RNAi) is expressed in the cell of the plant or plant component, thereby reducing expression of the tobacco nicotine demethylase in the plant or plant component. Ideally, a transgene encoding an antisense sequence of a tobacco nicotine demethylase or an RNA molecule capable of inducing RNA interference (RNAi) is constitutively or inducibly expressed, e.g., in a tissue-specific, cell-specific or organ-specific manner.

Another aspect of the invention features another method of reducing expression of a tobacco nicotine demethylase in a plant or plant component. The method comprises the following steps: (a) introducing a transgene into a plant cell to produce a transformed plant cell, said transgene encoding a dominant negatively regulated gene product of a tobacco nicotine demethylase operably linked to a promoter functional in the plant cell; and (b) regenerating a plant or plant component from the transformed plant cell, wherein the dominant negative gene product of tobacco nicotine demethylase is expressed in the cell of the plant or plant component, thereby reducing expression of tobacco nicotine demethylase in the plant or plant component. In a particular embodiment of this aspect of the invention, the transgene encoding the dominant negative gene product is constitutively or inducibly expressed, for example, in a tissue-specific, cell-specific or organ-specific manner.

Another aspect of the invention features another method of reducing the expression or enzymatic activity of a tobacco nicotine demethylase in a plant cell. The method comprises reducing the level of endogenous tobacco nicotine demethylase, or its enzymatic activity, in a plant cell. Desirably, the plant cell is from a dicotyledonous plant, a plant of the family Solanaceae, or a tobacco plant species. In desirable embodiments of this aspect, reducing the endogenous level of tobacco nicotine demethylase comprises expressing in a plant cell a transgene encoding an antisense nucleic acid molecule encoding a tobacco nicotine demethylase or an RNA molecule capable of inducing RNA interference (RNAi), or expressing in a plant cell a double-stranded RNA molecule encoding a tobacco nicotine demethylase. Desirably, the double stranded RNA is a dna corresponding to SEQ id no:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or a fragment thereof. In another embodiment, reducing the level of an endogenous tobacco nicotine demethylase comprises co-inhibiting the endogenous tobacco nicotine demethylase in a plant cell or comprises expressing a dominant negative gene product in a plant cell. In particular, the dominant negative gene product can include a nucleotide sequence encoding SEQ ID NO: 2 in a mutated form of the amino acid sequence of seq id No. 2.

In other desirable embodiments of this aspect of the invention, the endogenous tobacco nicotine demethylase is comprised in a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 in a gene of the amino acid sequence of seq id No. 2. In other desirable embodiments, reducing the level of expression of an endogenous tobacco nicotine demethylase comprises a deletion in, or an insertion in, a gene encoding a tobacco nicotine demethylase. Reduced expression may occur at the transcriptional level, at the translational level, or at the post-translational level.

In another aspect of the invention features a method of identifying a compound that alters expression of a tobacco nicotine demethylase in a cell. The method comprises the following steps: (a) providing a cell comprising a gene encoding a tobacco nicotine demethylase; (b) applying a candidate compound to a cell; and (c) measuring the expression of a gene encoding a tobacco nicotine demethylase, wherein an increase or decrease in expression relative to an untreated control sample is indicative of a compound altering the expression of a tobacco nicotine demethylase.

In desirable embodiments of the method, the gene of part (a) encodes a tobacco nicotine demethylase that hybridizes with SEQ ID NO: 2 has at least 70% identity. Desirably, the compound reduces or increases the expression of a gene encoding the tobacco nicotine demethylase.

In another aspect, the invention features another method of identifying a compound that alters the activity of a tobacco nicotine demethylase in a cell. The method comprises the following steps: (a) providing a cell expressing a gene encoding a tobacco nicotine demethylase; (b) applying a candidate compound to a cell; and (c) measuring the activity of the tobacco nicotine demethylase, wherein an increase or decrease in activity relative to an untreated control sample is indicative of a compound altering the activity of the tobacco nicotine demethylase. In desirable embodiments of this aspect of the invention, the gene of step (a) encodes a tobacco nicotine demethylase that hybridizes with the nucleic acid sequence of SEQ ID NO: 2 has at least 70% identity. Desirably, the compound reduces or increases the activity of tobacco nicotine demethylase.

Another aspect of the invention features a processed tobacco plant or plant component that includes (i) a reduced level of nicotine demethylase or (ii) a nicotine demethylase with altered enzymatic activity and a reduced amount of nitrosamines. Desirably, the plant component is tobacco leaf or tobacco stalk. In desirable embodiments, the nitrosamine is nornicotine, and desirably the nornicotine is present in an amount less than 5mg/g, 4.5mg/g, 4.0mg/g, 3.5mg/g, 3.0mg/g, more desirably less than 2.5mg/g, 2.0mg/g, 1.5mg/g, 1.0mg/g, more desirably less than 750 μ g/g, 500 μ g/g, 250 μ g/g, 100 μ g/g, even more desirably less than 75 μ g/g, 50 μ g/g, 25 μ g/g, 10 μ g/g, 7.0 μ g/g, 5.0 μ g/g, 4.0 μ g/g, and even more desirably less than 2.0 μ g/g, 1.0 μ g/g, 0.5 μ g/g, 0.4 μ g/g, 0.2 μ g/g, 0.1 μ g/g, 0.05 μ g/g, or 0.01 μ g/g, or wherein the percentage of secondary alkaloids relative to the total alkaloid content therein is less than 90%, 70%, 50%, 30%, 10%, desirably less than 5%, 4%, 3%, 2%, 1.5%, 1%, and more desirably less than 0.75%, 0.5%, 0.25%, or 0.1%. In another desirable embodiment, the nitrosamine is N '-nitrosonornicotine (NNN), and the amount of N' -NNN is desirably less than 5mg/g, 4.5mg/g, 4.0mg/g, 3.5mg/g, 3.0mg/g, more desirably less than 2.5mg/g, 2.0mg/g, 1.5mg/g, 1.0mg/g, more desirably less than 750 μ g/g, 500 μ g/g, 250 μ g/g, 100 μ g/g, even more desirably less than 75 μ g/g, 50 μ g/g, 25 μ g/g, 10 μ g/g, 7.0 μ g/g, 5.0 μ g/g, 4.0 μ g/g, and even more desirably less than 2.0 μ g/g, 1.0 μ g/g, 0.5 μ g/g, 0.4 μ g/g, 0.2 μ g/g, 0.1 μ g/g, 0.05 μ g/g, or 0.01 μ g/g, or wherein the percentage of secondary alkaloids relative to the total alkaloid content it comprises is less than 90%, 70%, 50%, 30%, 10%, ideally less than 5%, 4%, 3%, 2%, 1.5%, 1%, and more ideally less than 0.75%, 0.5%, 0.25%, or 0.1%. In another desirable embodiment of this aspect of the invention, the processed tobacco plant or plant component is dark tobacco (dark tobaco), Burley tobaco, smoke-cured tobaco, dark air-cured tobaco, or oriental tobacco (original tobaco).

In addition, the processed tobacco plants or plant components of the invention desirably include a recombinant nicotine demethylase gene, such as a gene comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 3, or a fragment thereof. Ideally, expression of the endogenous nicotine demethylase gene is silenced in the processed tobacco plant or plant component.

Another aspect of the invention features a tobacco product comprising a processed tobacco plant or plant component that includes (i) reduced expression of a nicotine demethylase or (ii) a nicotine demethylase with altered activity, and a reduced amount of nitrosamines. Desirably, the tobacco product is smokeless tobacco, wet or dry snuff, chewing tobacco, cigarette, cigar, cigarillo, pipe tobacao or bidis. In particular, the tobacco product of this aspect of the invention may comprise dark tobacco, ground tobacco (milled tobaco), or include flavoring ingredients.

The invention also features methods of making tobacco products, such as smokeless tobacco products, that include (i) reduced expression of a nicotine demethylase or (ii) a nicotine demethylase having altered (e.g., reduced) enzymatic activity, and a reduced amount of a nitrosamine. The method comprises providing a processed tobacco plant or plant component comprising (i) a reduced level of nicotine demethylase or (ii) a nicotine demethylase having altered enzymatic activity and a reduced amount of nitrosamines, and preparing a tobacco product from said processed tobacco plant or plant component.

1. An isolated nucleic acid molecule encoding a nicotine demethylase, wherein the nucleic acid molecule comprises (a) the amino acid sequence of SEQ id no:1, or (b) a sequence identical to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or (c) at least 200 consecutive base pairs identical to 200 consecutive base pairs of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 is at least 20 consecutive base pairs identical.

2. The isolated nucleic acid molecule of item 1, wherein said nucleic acid molecule is DNA.

3. The isolated nucleic acid molecule of item 1, wherein the nucleic acid molecule comprises SEQ ID NO: 1.

4. The isolated nucleic acid molecule of item 1, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

5. The isolated nucleic acid molecule of item 1, wherein said isolated nucleic acid molecule is operably linked to a promoter functional in a plant cell.

6. The isolated nucleic acid molecule of item 1, comprising a sequence that hybridizes under stringent conditions to SEQ ID NO: 6 or a fragment thereof, which fragment drives transcription.

7. The isolated nucleic acid molecule of item 6, wherein the promoter is (i) induced after treatment with ethylene or during senescence; and (ii) a polypeptide comprising (a) SEQ ID NO:1, or (b) binds to a polypeptide consisting of SEQ ID NO:1, at least 200 consecutive base pairs that are identical in sequence to 200 consecutive base pairs of the sequence defined by base pairs 1-2009, or (c) a sequence that is identical in sequence to SEQ ID NO:1, base pairs 1-2009 from the sequence set forth in the 20 contiguous base pair portions of the same 20 base pair nucleotide portion.

8. The isolated nucleic acid molecule of item 1, comprising a nucleotide sequence that is identical to SEQ ID NO: 6 has a nucleotide sequence of 50% or greater sequence identity.

9. The isolated nucleic acid molecule of item 1 or 8, wherein said nucleic acid molecule is induced after treatment with ethylene or during senescence.

10. The isolated nucleic acid molecule of item 1 or 8, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6.

11. The isolated nucleic acid molecule of item 1 or 8, wherein the nucleic acid molecule comprises a sequence derivable from SEQ ID NO: 6, wherein the fragment drives transcription of a heterologous gene.

12. The isolated nucleic acid molecule of item 8 operably linked to a heterologous nucleic acid sequence.

13. The isolated nucleic acid molecule of item 1, comprising an intron that hybridizes under stringent conditions to the complement of SEQ id no: 5 or a fragment thereof.

14. The isolated nucleic acid molecule of item 13, wherein the intron comprises (a) the nucleotide sequence of SEQ ID NO:1, or (b) differs from the sequence defined by SEQ ID NO:1 base pair 2950-: 1 base pair 2950-.

15. The isolated nucleic acid of item 1, wherein the nucleic acid molecule comprises a nucleotide sequence that is identical to SEQ ID NO: 5 or a fragment thereof has a nucleotide sequence of 50% or greater sequence identity.

16. The isolated nucleic acid molecule of item 1 or 13, wherein the nucleic acid molecule comprises SEQ ID NO: 5.

17. The isolated nucleic acid molecule of item 1 or 13, wherein said nucleic acid molecule comprises a sequence derivable from SEQ ID NO: 5 the fragment obtained.

18. The isolated nucleic acid molecule of item 13 operably linked to a heterologous nucleic acid sequence.

19. The isolated nucleic acid molecule of item 1, comprising an untranslated region that hybridizes, under stringent conditions, to the complement of seq id NO: 7, or a fragment thereof.

20. The isolated nucleic acid molecule of item 19, wherein the untranslated region comprises (a) the amino acid sequence of SEQ ID NO:1, or (b) differs from the base pair 4563-6347 of SEQ ID NO:1 base pair 4563-6347 or (c) at least 200 consecutive base pairs identical in sequence to 200 consecutive base pairs of the sequence defined by SEQ ID NO:1 base pair 4563-6347 proposes a 20 base pair nucleotide portion identical to a 20 base pair portion of the sequence set forth in base pairs 20 contiguous.

21. The isolated nucleic acid molecule of item 1, wherein the nucleic acid molecule comprises a nucleotide sequence that is identical to SEQ ID NO: 7 has a nucleotide sequence of 50% or greater sequence identity.

22. The isolated nucleic acid molecule of item 1 or 19, wherein the nucleic acid molecule comprises SEQ ID NO: 7.

23. The isolated nucleic acid molecule of item 1 or 19, wherein said nucleic acid molecule comprises a sequence derivable from SEQ ID NO: 7, and (c) the fragment obtained.

24. The isolated nucleic acid molecule of item 19 operably linked to a heterologous nucleic acid sequence.

25. A tobacco plant having reduced expression or altered enzymatic activity of a nicotine demethylase encoded by the nucleic acid molecule of item 1, wherein said reduced expression or altered enzymatic activity reduces the level of nornicotine or N' -nitrosonornicotine in said plant.

26. The tobacco plant of item 25, wherein said nicotine demethylase comprises the amino acid sequence of SEQ ID NO: 2.

27. The tobacco plant of item 25, wherein said plant is a transgenic plant.

28. The tobacco plant of item 27, wherein said transgenic plant comprises a transgene that, when expressed in said transgenic plant, silences gene expression of an endogenous tobacco nicotine demethylase.

29. The tobacco plant of item 27, wherein said transgenic plant comprises a transgene that expresses an antisense molecule of a tobacco nicotine demethylase.

30. The tobacco plant of item 27, wherein said transgenic plant comprises a transgene that encodes a double stranded RNA molecule of a tobacco nicotine demethylase.

31. The tobacco plant of item 27, wherein said transgenic plant comprises a transgene that, when expressed in said transgenic plant, co-inhibits expression of tobacco nicotine demethylase.

32. The tobacco plant of item 27, wherein said transgenic plant comprises a transgene encoding a dominant negative gene product.

33. The tobacco plant of item 32, wherein the dominant negative gene product comprises a nucleotide sequence encoding SEQ id no: 2 in a mutated form of the amino acid sequence of seq id No. 2.

34. The tobacco plant of item 33, wherein said plant comprises a polynucleotide encoding SEQ ID NO: 2 in a gene of the amino acid sequence of seq id No. 2.

35. The tobacco plant of item 27, wherein said plant comprises a deletion in a gene encoding a tobacco nicotine demethylase.

36. The plant component of the tobacco plant of item 25.

37. The plant component of item 36, wherein the plant component is a tobacco leaf, stem, or seed.

38. The plant component of item 37, wherein said leaves are processed.

39. The plant component of item 36, wherein the nornicotine content is less than 4.0 μ g/g.

40. The plant component of item 36, wherein the content of secondary alkaloids is less than 3% relative to the total alkaloid content.

41. The plant component of item 36, wherein the content of N' -nitrosonornicotine is less than 4.0 μ g/g.

42. A tobacco product comprising the plant component of any one of items 36-41.

43. Seed from the tobacco plant of item 25.

44. A method of producing a tobacco nicotine demethylase, said method comprising the steps of:

(a) providing a cell transformed with the isolated nucleic acid molecule of item 1;

(b) culturing the transformed cell under conditions in which the isolated nucleic acid molecule is expressed; and

(c) recovering the tobacco nicotine demethylase.

45. A recombinant tobacco nicotine demethylase prepared in accordance with the method of item 44.

46. A method of isolating a tobacco nicotine demethylase, or fragment thereof, said method comprising the steps of:

(a) contacting the nucleic acid molecule of item 1 with a preparation of nucleic acid from a plant cell under hybridization conditions that provide for detection of a nucleic acid sequence having at least 70% or greater sequence identity to the nucleic acid molecule of item 1; and

(b) isolating the hybridized nucleic acid sequence.

47. A method of isolating a tobacco nicotine demethylase, or fragment thereof, said method comprising the steps of:

(a) providing a sample of plant cell DNA;

(b) providing a pair of oligonucleotides having sequence identity to a region of the nucleic acid molecule of item 1;

(c) contacting the oligonucleotide pair with said plant cell DNA under conditions suitable for polymerase chain reaction-mediated DNA amplification; and

(d) isolating the amplified tobacco nicotine demethylase or fragment thereof.

48. The method of item 47, wherein said amplifying step is performed using a cDNA sample prepared from a plant cell.

49. The method of item 47, wherein said tobacco nicotine demethylase encodes a polypeptide that hybridizes to a sequence selected from the group consisting of SEQ ID NO: 2 has at least 70% identity.

Definition of

"enzymatic activity" is meant to include, but is not limited to, demethylation, hydroxylation, epoxidation, N-oxidation, thiooxidation, N-, S-, and O-dealkylation, desulfurization, deamination, and reduction of azo, nitro, and N-oxide and other such enzymatically reactive chemical groups. Altered enzymatic activity refers to a reduction in enzymatic activity (e.g., a reduced enzymatic activity of a tobacco nicotine demethylase) by at least 10-20%, preferably at least 25-50%, and more preferably at least 55-95% or more, relative to the activity of a control enzyme (e.g., a wild-type tobacco plant nicotine demethylase). The activity of tobacco nicotine demethylase may be determined using standard methods in the art, such as measuring the demethylation of radioactive nicotine by yeast-expressed microsomes as described herein.

The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer, either sense or antisense, in either single-or double-stranded form, and unless otherwise defined, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes its complement. The terms "operably linked," "in operable combination," and "in operable order" refer to a functional linkage between a nucleic acid expression control sequence (such as a promoter, a signal sequence, or an array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects the transcription and/or translation of the nucleic acid corresponding to the second sequence.

The term "recombinant" when used with respect to a cell indicates that the cell replicates a heterologous nucleic acid, expresses the nucleic acid or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells may express genes or gene fragments in sense or antisense form or RNA molecules capable of inducing RNA interference (RNAi) that are not found in the native (non-recombinant) form of the cell. Recombinant cells may also express genes found in the native form of the cell, but where the genes are modified in an artificial manner and reintroduced into the cell.

A "structural gene" is a portion of a gene that includes a DNA segment encoding a protein, polypeptide, or portion thereof, and does not include, for example, the 5 'sequence or 3' UTR that drives transcription initiation. The structural gene may alternatively encode a nontranslatable product. The structural gene may be a gene normally found in the cell or a gene not normally found in the cell or the cellular location into which it is introduced, in which case it is referred to as a "heterologous gene". The heterologous gene may be derived in whole or in part from any source known in the art, including bacterial genomes or episomes (episomes), eukaryotic nuclear or plasmid DNA, cDNA, viral DNA, or chemically synthesized DNA. The structural gene may comprise one or more modifications that may affect the biological activity or a characteristic thereof, the biological activity or chemical structure of the expression product, the rate of expression or the manner in which expression is controlled. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides.

The structural gene may consist of an uninterrupted coding sequence or it may comprise one or more introns joined by suitable splice junctions. The structural gene may be translatable or nontranslatable, including antisense or RNA molecules capable of inducing RNA interference (RNAi). The structural gene may be a composition of fragments from multiple sources and from multiple gene sequences (naturally occurring or synthetic, where synthetic refers to chemically synthesized DNA).

As used herein with respect to nucleic acid sequences, "exon" refers to the portion of a nucleic acid sequence of a gene, wherein the nucleic acid sequence of the exon encodes at least one amino acid of the gene product. Exons are typically adjacent to non-coding DNA segments such as introns. Ideally, the exon encodes SEQ ID NO: 2, such as SEQ ID NO: 2 sequence of amino acids 1-313 and/or 314-517.

As used herein with respect to nucleic acid sequences, "intron" refers to the non-coding region of a gene that flanks a coding region. Introns are typically non-coding regions of a gene that are transcribed into RNA molecules, but are then excised by RNA splicing in the process of producing messenger RNA or other functional structural RNA. Ideally, the intron comprises SEQ ID NO: 5, or a fragment thereof.

As used herein with respect to nucleic acid sequences, "3' UTR" refers to a non-coding nucleic acid sequence adjacent to a stop codon for an exon. Desirably, the 3' UTR comprises SEQ ID NO: 7, or a fragment thereof.

"derived from" is used to mean taken from, obtained from, received from, found from, replicated from, or inherited from a source (chemical and/or biological). Derivatives may be prepared by chemical or biological manipulation of the original source (including, but not limited to, substitution, addition, insertion, deletion, extraction, isolation, mutation, and replication).

"chemical synthesis" in relation to a DNA sequence refers to the assembly of the parts that make up the nucleotides in vitro. Manual chemical synthesis of DNA can be accomplished using well-known methods (carothers,Methodology of DNA and RNA Sequencing(1983), Weissman (ed.), Praeger Publishers, New York, Chapter I); automated chemical synthesis can be performed using one of many commercially available machines.

The optimized alignment of sequences to be compared may be determined, for example, by Smith and Waterman, adv.appl.math.2: 482(1981), Needleman and Wunsch, j.mol.biol.48: 443(1970) by Pearson and Lipman proc.natl.acad.sci. (u.s.a.) 85: 2444(1988), by Computer execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin genetic software package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, 1990) was obtained from several sources, including the National Center for Biological Information (NCBI, Bethesda, Md.) and on the Internet, in combination with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. It can be evaluated on http:// www.ncbi.nlm.nih.gov/BLAST/. A description of how to use this program to determine sequence identity is available in http:// www.ncbi.nlm.nih.gov/BLAST/BLAST help.

As used in amino acid sequences and herein, the term "substantial amino acid identity" or "substantial amino acid sequence identity" refers to a characteristic of a polypeptide, wherein the peptide comprises a sequence identical to a sequence of SEQ ID NOS: 2 and/or 4, preferably 80% amino acid sequence identity, more preferably 90% amino acid sequence identity, and most preferably at least 99 to 100% sequence identity. Ideally, for nicotine demethylase, sequence comparisons are ideally used for comparison of the cytochrome p450 motif (GXRXCX (G/A); SEQ ID NO: 29) to the region of the termination codon of the translated peptide.

As used in nucleic acid sequences and herein, the term "substantial nucleic acid identity" or "substantial nucleic acid sequence identity" refers to a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that is identical to a sequence of SEQ ID NOS: 1, 3, 5, 6, and/or 7, having at least 50%, preferably 60%, 65%, 70%, or 75% sequence identity, more preferably 81% or 91% nucleic acid sequence identity, and most preferably at least 95%, 99%, or even 100% sequence identity. Ideally, for nicotine demethylase nucleic acid sequences, the comparison is ideal for comparison of the region encoding the cytochrome p450 motif (GXRXCX (G/A); SEQ ID NO: 29) to the stop codon of the translated peptide.

Another indication that nucleotide sequences are substantially identical is whether two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions for selection of a particular sequence are about 5 ℃ to about 20 ℃, typically about 10 ℃ to about 15 ℃ below the thermal melting point (Tm) at a defined ionic strength and pH. The Tm is the temperature (at defined ionic strength and pH) at which 50% of the target sequence hybridizes to a matching probe. Typically, stringent conditions will be those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60 ℃. For example, in standard DNA hybridization methods, stringent conditions will include an initial wash in 6XSSC at 42 ℃ followed by one or more additional washes in 0.2XSSC at a temperature of at least about 55 ℃, typically about 60 ℃, and usually about 65 ℃.

For the purposes of the present invention, nucleotide sequences are also substantially identical when they encode substantially identical polypeptides and/or proteins. Thus, when one nucleic acid sequence encodes a polypeptide that is substantially identical to a Second nucleic acid sequence, the two nucleic acid sequences are substantially identical, even though they do not hybridize under stringent conditions due to the degeneracy permitted by the genetic code (see, Darnell et al (1990) Molecular Cell Biology, Second edition scientific American Books W.H.Freeman and Company New York for an expansion of code deputy and the genetic code). Protein purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample followed by visualization by staining. For some purposes, high resolution may be required, and HPLC or similar means for purification may be used.

By "specifically binds" or "specifically recognizes" an antibody to a tobacco nicotine demethylase is meant an antibody to a tobacco nicotine demethylase that has increased affinity relative to an equivalent amount of any other protein. For example, specifically binds to a polypeptide comprising SEQ id no: 2 desirably has an affinity for its antigen that is at least 2-fold, 5-fold, 10-fold, 30-fold, or 100-fold greater than an equivalent amount of any other antigen, including related antigens. Binding of the antibody to an antigen, e.g., tobacco nicotine demethylase, can be determined by a number of methods standard in the art, e.g., western blot analysis, ELISA, or co-immunoprecipitation.

As used herein, the term "vector" refers to a nucleic acid molecule used to deliver DNA fragments to a cell. The vector may act to replicate the DNA and may propagate independently in the host cell. The term "vehicle" is sometimes used interchangeably with "carrier". As used herein, the term "expression vector" refers to a recombinant DNA molecule comprising a desired coding sequence and appropriate nucleic acid sequences necessary for expression of an operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes generally include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Desirably, the promoter comprises SEQ ID NO: 6, or a fragment thereof. Furthermore, it is desirable to have a sequence identical to SEQ ID NO: 6 has at least 50%, 60%, 75%, 80%, 90%, 95%, or even 99% sequence identity and drives transcription. Eukaryotic cells are known to use promoters, enhancers and terminators as well as polyadenylation signals, such as SEQ ID NO: 7, 3' UTR sequence. In certain instances, it has been observed that plant expression vectors require plant-derived introns, such as those having the sequence of SEQ ID NO: 5 to have stable expression. Likewise, SEQ ID NO: 5, or any other intron having a suitable RNA splice junction may be used as further described herein.

For the purpose of regenerating a whole genetically engineered plant having roots, the nucleic acid may be inserted into a plant cell, for example by any technique such as in vivo inoculation or by any known in vitro tissue culture technique to produce a transformed plant cell that can be regenerated into a whole plant. Thus, for example, insertion of plant cells may be performed by in vitro inoculation with pathogenic or non-pathogenic a. Other such tissue culture techniques may also be used.

"plant tissue", "plant components" or "plant cells" include differentiated and undifferentiated tissues of plants, including, but not limited to, cultured roots, stems, leaves, pollen, seeds, tumor tissue, and various forms of cells such as single cell, protoplast, embryo, and callus tissue. The plant tissue may be in a plant or present as an organ, tissue or cell culture.

As used herein, "plant cell" includes plant cells in plants and cultured plant cells and protoplasts. "cDNA" or "complementary DNA" generally refers to a single-stranded DNA molecule having a nucleotide sequence that is complementary to either an unprocessed RNA molecule that contains introns, or a processed mRNA that lacks introns. The cDNA is formed by the action of the enzyme reverse transcriptase on the RNA template.

As used herein, "tobacco" includes smoke, Virginia (Virginia), burley, dark, oriental, and other types of plants in the nicotiana genus. Seeds of nicotiana are readily commercially available in the form of tobacco.

"article" or "tobacco product" includes products such as wet and dry snuff, chewing tobacco, cigarettes, cigars, cigarillos, pipe tobacao, bidis and similar tobacco-derived products.

By "gene silencing" is meant a reduction in the level of gene expression (e.g., expression of a gene encoding a tobacco nicotine demethylase) by at least 30-50%, preferably at least 50-80%, and more preferably at least 80-95% or greater, relative to the level of a control plant (e.g., a wild-type tobacco plant). Such reduction in expression levels can be accomplished by generation of the mutated gene using standard methods known in the art including, but not limited to, RNA interference, triple-strand interference, ribozymes, homologous recombination, virus-induced gene silencing, antisense and cosuppression techniques, expression of dominant negative gene products, or by using standard mutagenesis techniques, such as those described herein. The level of tobacco nicotine demethylase polypeptide or transcript, or both, is monitored according to any standard technique, including, but not limited to, northern blot, ribonuclease protection, or immunoblot.

As used herein, "tobacco nicotine demethylase" or "nicotine demethylase" refers to a polypeptide that substantially corresponds to the amino acid sequence of seq id NO: 2. Ideally, the tobacco nicotine demethylase is capable of converting nicotine (C)₁₀H₁₄N₂Also known as 3- (1-methyl-2-pyrrolidinyl) pyridine) to nornicotine (C)₉H₁₂N₂). As described herein, the activity of tobacco nicotine demethylase can be assessed using methods standard in the art, such as demethylation of radioactive nicotine by measurement of yeast expressed microsomes.

By "fragment" or "portion" of the tobacco nicotine demethylase amino acid sequence is meant the amino acid sequence of SEQ ID NO: 2, e.g., at least 20, 15, 30, 50, 75, 100, 250, 300, 400, or 500 consecutive amino acids of the amino acid sequence of seq id No. 2. Exemplary ideal fragments are SEQ ID NO: 2 and amino acids 1-313 of the sequence of SEQ ID NO: 2, amino acid 314-. Further, with respect to fragments or portions of the tobacco nicotine demethylase nucleic acid sequence, desirable fragments include the amino acid sequence of SEQ ID NO:1, at least 100, 250, 500, 750, 1000, or 1500 consecutive nucleic acids of the nucleic acid sequence of 1. Exemplary ideal fragments are SEQ ID NO:1, nucleic acids 1-2009, 2010-2949, 2950-3946, 3947-4562, 4563-6347 and 4731-6347. Other desirable fragments include SEQ ID NOs: 5, nucleic acids 1-100, 101-250, 251-500, 501-750, and 751-998, SEQ ID NO: 6, 1-398, 1-1400, 1401-2009, 1840-2009, 1940-2009, 399-1240, and 1241-2009, and SEQ ID NO: 7 nucleic acids 1-100, 101-250, 251-500, 501-750, 751-1000, 1001-1250, 1251-1500 and 1501-1786.

By "substantially pure polypeptide" is meant a tobacco nicotine demethylase that has been isolated from most of the components with which it naturally accompanies; however, such other proteins, found in the microsomal fraction associated with the preparation, having a nicotine demethylase activity of at least 8.3pKat/mg protein, 9pKat/mg protein, 9.5pKat/mg protein, 10pKat/mg protein, 10.5pKat/mg, or 10.8pKat/mg protein, are also considered substantially pure polypeptides. Typically, the polypeptide is substantially pure when at least 60% by weight of the proteins and naturally occurring organic molecules with which it is naturally associated are removed. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight and most preferably at least 99% by weight of tobacco nicotine demethylase. Can be obtained, for example, by extraction from natural sources (e.g., tobacco plant cells); by expression of a recombinant nucleic acid encoding a tobacco nicotine demethylase; or by chemical synthesis of proteins to obtain a substantially pure tobacco nicotine demethylase. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By "isolated nucleic acid molecule" is meant a nucleic acid sequence with the nucleic acid sequences flanking the nucleic acid molecule sequences naturally located in the genome of the organism removed.

By "transformed cell" is meant a cell into which (or in an ancestor thereof) a DNA molecule, e.g., a DNA molecule encoding a tobacco nicotine demethylase, has been introduced by recombinant DNA techniques.

As provided herein, the terms "cytochrome p 450" and "p 450" are used interchangeably.

Brief Description of Drawings

FIG. 1 is a schematic representation of the genomic structure of a tobacco nicotine demethylase gene.

FIGS. 2-1 to 2-3 are the genomic tobacco nicotine demethylase nucleic acid sequence (SEQ ID NO: 1) and its translation product (SEQ ID NO: 2).

FIG. 3 is the nucleic acid sequence (SEQ ID NO: 3) (also referred to as D121-AA8) of the tobacco nicotine demethylase coding region and its translation product (SEQ ID NO: 4).

FIG. 4 is the nucleic acid sequence of an intron present in the tobacco nicotine demethylase genomic sequence (SEQ ID NO: 5).

FIG. 5 is a nucleic acid sequence of the promoter region of tobacco nicotine demethylase (SEQ ID NO: 6).

FIG. 6 is a nucleic acid sequence of the 3' UTR of the tobacco nicotine demethylase gene (SEQ ID NO: 7).

Detailed Description

Identification of genomic clones encoding tobacco nicotine demethylase

According to the present invention, RNA was extracted from the nicotiana tissue of the transformant (converter) and non-transformant nicotiana lines. The extracted RNA is then used to generate cDNA. Next, two strategies are used to generate the nucleic acid sequences of the invention.

In the first strategy, poly a-rich RNA is extracted from plant tissue and cDNA is prepared by reverse transcription PCR. Next, the single stranded cDNA was used to generate a p450 specific PCR population using degenerate primers plus oligo d (T) reverse primers. Primer design is based on highly conserved motifs of other plant cytochrome p450 gene sequences. Examples of specific degenerate primers are set forth in FIG. 1 in the publications of U.S. Pat. Nos. 2004/0103449A1 and 2004/0111759A1, which are incorporated herein by reference. The sequence of the fragment from the plasmid containing the insert of the appropriate size was further analyzed. Inserts of these sizes are typically from about 300 to about 800 nucleotides, depending on the primer used.

In the second strategy, the cDNA library was initially constructed. The cDNA in the plasmid was used to generate p 450-specific PCR populations using degenerate primers plus T7 primer on the plasmid as reverse primer. As in the first strategy, the sequence of the fragment from the plasmid containing the appropriately sized insert was further analyzed.

Nicotiana plant lines (transformants) known to produce high levels of nornicotine and plant lines with low levels of nornicotine can be used as starting materials. The leaves can then be removed from the plant and treated with ethylene to activate the p450 enzyme activity as defined herein. Total RNA was extracted using techniques known in the art. Subsequently, PCR with oligo d (T) primers (RT-PCR) can be used to generate cDNA fragments. Next, a cDNA library can be constructed as described more fully in the examples herein.

The p450 type enzyme conserved region was used as a template for degenerate primers. The p450 specific band was amplified by PCR using degenerate primers. Bands indicative of p 450-like enzymes were identified by DNA sequencing. PCR fragments are characterized using BLAST searches, alignments or other tools to identify suitable candidates.

Sequence information from the identified fragments was used to develop PCR primers. These primers were combined with plasmid primers in a cDNA library for cloning the full-length p450 gene. Large-Scale Southern reverse analysis was performed to examine all fragments obtainedDifferential expression of clones and in some cases full-length clones. In this aspect of the invention, these large scale reverse Southern assays can be performed to screen all cloned inserts using labeled total cDNAs from different tissues as probes to hybridize with the cloned DNA fragments. Will also be non-radioactive and radioactive (P)³²) Northern blotting was used to characterize the cloned p450 fragment and full-length clones.

Peptide-specific antibodies were prepared by deriving their amino acid sequences and selecting peptide regions that were antigenic and unique relative to other clones. Rabbit antibodies were prepared to synthesize peptides conjugated to carrier proteins. Using these antibodies, western blot analysis or other immunological methods were performed on plant tissues. In addition, peptide-specific antibodies against several full-length clones were prepared by deriving their amino acid sequences and selecting peptide regions that are potentially antigenic and unique relative to other clones. Rabbit antibodies were prepared to synthesize peptides conjugated to carrier proteins. Using these antibodies, western blot analysis was performed.

Down-regulation of tobacco nicotine demethylase

Plants with reduced expression of tobacco nicotine demethylase were generated according to standard gene silencing methods. (for review see, Arndt and Rank, Genome 40: 785-. Reduced expression of a tobacco nicotine demethylase gene may be achieved using, for example, RNA interference (RNAi) (Smith et al, Nature 407: 319; 320, 2000; Fire et al, Nature 391: 306-; virus-induced gene silencing ("VIGS") (Baulcombe, Current Opinions in Plant Biology, 2: 109-113, 1999; Cogoni and Macino, Genes Dev 10: 638-643, 2000; Ngelbrecht et al, PNAS 91: 10502-10506, 1994); silencing of the target gene by transmission of the Plant endogenous gene in sense orientation (Jorgensen et al, Plant Mol Biol 31: 957-; expression of an antisense gene; homologous Recombination (Ohl et al, homologus Recombination and Gene cloning in Plants (Kluwer, Dordrecht, the Netherlands, 1994); the Cre/lox system (Qin et al, PNAS 91: 1706-1710, 1994; koshinsky et al, The Plant Journal 23: 715-addition 722, 2000; chou, et al, plant and Animal Genome VIIConference abstracts. san Diego, CA, 1 month 17-211999); gene capture and T-DNA markers (Burnstet al, Genes Dev.8: 1087-; spradling, et al, PNAS 92: 10824-; skrarnes et al, Bio/Technology 8, 827-831, 1990; sundaresan, et al, Genes Dev.9: 1797-; and any other possible gene silencing system, which is available in the silencing region, which results in the down-regulation of the expression of tobacco nicotine demethylase or a reduction in its enzymatic activity.

RNA interference

RNA interference ("RNAi") is an applicable method commonly used to induce efficient and specific post-translational gene silencing in many organisms, including plants (see, e.g., Bosher et al, nat. cell biol. 2: E31-36, 2000; and Tavernarakis et al, nat. genetics 24: 180-. RNAi involves the introduction of RNA with partial or full-length double-stranded characteristics into a cell or into the extracellular environment. Inhibition is specific in that a nucleotide sequence from a portion of a gene of interest (e.g., tobacco nicotine demethylase) is selected to produce an inhibitory RNA. The selected portion will typically comprise an exon of the target gene, but the selected portion may also comprise untranslated regions (UTRs), as well as introns (e.g., of SEQ ID NO: 5 or 7).

For example, to construct a transformation vector that produces RNAs capable of forming duplexes, two tobacco nicotine demethylase nucleic acid sequences, one in sense and one in antisense orientation, may be operably linked and placed under the control of a strong viral promoter, such as CaMV 35S, or a promoter isolated from Cassava Brown Streak Virus (CBSV). However, endogenous promoters are used, such as those having SEQ ID NO: 6, or a fragment thereof that drives transcription may also be desirable. The tobacco nicotine demethylase nucleic acid sequence included in such a construct is desirably at least 25 nucleotides in length, but may include sequences that include up to the full-length tobacco nicotine demethylase gene.

Constructs producing RNAs capable of forming duplexes can be introduced into the genome of plants such as tobacco plants by Agrobacterium-mediated transformation (Chuang et al, Proc. Natl. Acad. Sci. USA 97: 4985-4990, 2000), resulting in specific and heritable genetic interference in tobacco nicotine demethylase. The double-stranded RNA can also be introduced directly into the cell (i.e., intracellularly) or extracellularly, for example, by bathing the seed, seedling, or plant in a solution comprising the double-stranded RNA.

The RNAi can provide partial or complete loss of function of the target gene depending on the dose of double-stranded RNA species delivered. A reduction or loss of gene expression can be obtained in at least 99% of the cells of interest. In general, lower doses of injected material and longer times after administration of dsRNA result in inhibition of a smaller fraction of cells.

RNA used in RNAi can include one or more strands of polymerized ribonucleotides; it may include changes to the phosphate-sugar backbone or the nucleoside. The double-stranded structure may be formed by a single self-complementary RNA strand or by two complementary RNA strands, and RNA duplex formation may begin inside or outside the cell. The RNA may be introduced in an amount that allows at least one copy to be delivered per cell. However, higher doses (e.g., at least 5,10, 100, 500, or 1000 copies per cell) of double-stranded material may produce more effective inhibition. Inhibition is sequence specific in that genetic inhibition is directed to nucleotide sequences corresponding to the duplex region of the RNA. For inhibition, RNA comprising a nucleotide sequence identical to a portion of the target gene is preferred. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence may also be effective for inhibition. Thus, sequence identity can be optimized by alignment algorithms known in the art and calculating percent differences between nucleotide sequences. Alternatively, the duplex region of the RNA may be functionally defined as a nucleotide sequence capable of hybridizing to a portion of the target gene transcript.

In addition, RNA for RNAi can be synthesized in vivo or in vitro. For example, endogenous RNA polymerase in the cell may mediate in vivo transcription, or cloned RNA polymerase may be used for in vivo or in vitro transcription. For transcription from transgenes or expressed constructs in vivo, the regulatory region may be used to translate one strand (or multiple strands) of the RNA.

Three-chain interference

Endogenous tobacco nicotine demethylase gene expression can also be down-regulated by targeting deoxyribonucleotide sequences complementary to regulatory regions (e.g., promoter or enhancer regions) of the tobacco nicotine demethylase gene to form a triple helix structure that prevents transcription of the tobacco nicotine demethylase gene in the target cell (generally, Helene, Anticancer Drug Des.6: 569-584, 1991; Helene et al, Ann.N.Y.Acad.Sci.660: 27-36, 1992; and Maher, Bioassays 14: 807, 1992).

The nucleic acid molecule used in triple helix formation for inhibition of transcription is preferably single-stranded and consists of deoxyribonucleotides. The base composition of these oligonucleotides should promote triple helix formation using the Hoogsteen base pairing rules, which generally require a substantial stretch of purine or pyrimidine bases to be present on one strand of the duplex. The nucleotide sequence may be pyrimidine-based, which will result in TAT and CGC triplets across the three related strands of the resulting triple helix. The pyrimidine-rich molecule provides base complementarity to the purine-rich region of the single strand of the duplex in a direction parallel to that strand. In addition, purine-rich nucleic acid molecules, such as a stretch of G residues, can be selected. These molecules will form a triple helix with the GC-rich pair of DNA duplexes, with most of the purine residues located on a single strand of the target duplex, resulting in a CGC triplet that passes through three strands in the triple helix.

Alternatively, the possible sequences that can be targeted for triple helix formation can be increased by creating "turn-around" nucleic acid molecules. The diverter molecules are synthesized in an alternating 5 '-3', 3 '-5' fashion such that they base pair with the first strand of the duplex and then base pair with the other strand, eliminating the necessity for a substantial stretch of purine or pyrimidine to be present on one strand of the duplex.

Ribonuclease

Ribonucleases are RNA molecules that function as enzymes and can be engineered to cleave other RNA molecules. Ribonucleases can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific site, thereby functionally inactivating the target RNA. In this process, ribonuclease itself is not consumed and can act catalytically to cleave multiple copies of an mRNA target molecule. Accordingly, ribonucleases can also be used as a tool to down-regulate the expression of tobacco nicotine demethylase. The design and use of target RNA-specific ribonucleases is described in Haseloff et al (Nature 334: 585-591, 1988). Preferably, the ribonuclease comprises at least about 20 contiguous nucleotides complementary to a target sequence (e.g., tobacco nicotine demethylase) on each side of the ribonuclease's active site.

In addition, ribonuclease sequences can also be included in antisense RNA to confer RNA-cleaving activity on the antisense RNA and thus increase the effectiveness of the antisense construct.

Homologous recombination

Gene replacement technology is another desirable method of down-regulating the expression of a given gene, such as tobacco nicotine demethylase. Gene replacement techniques are based on homologous recombination (see, Schnable et al, curr. opinions Plant biol.1: 123-129, 1998). The nucleic acid sequence of a target enzyme, such as tobacco nicotine demethylase, may be manipulated by mutagenesis (e.g., insertion, deletion, replication, or substitution) to reduce the function of the enzyme. Subsequently, the altered sequence can be introduced into the genome by homologous recombination to replace existing, for example wild-type genes (Puchta et al, Proc. Natl. Acad. Sci. USA 93: 5055-5060, 1996; and Kempin et al, Nature 389: 802-803, 1997).

Co-suppression

Another desirable method of silencing gene expression is co-suppression (also known as sense suppression). This technique, which involves the introduction of nucleic acids constructed in sense orientation, has shown a significant hindrance to transcription of the target gene (see, e.g., Napoliet al, Plant Cell, 2: 279-289, 1990 and Jorgensen et al, U.S. Pat. No. 5,034,323).

In general, sense suppression includes transcription of the introduced sequence. However, cosuppression may also occur when the introduced sequence itself comprises a non-coding sequence, but only an intron (e.g., the sequence of SEQ ID NO: 5) or an untranslated sequence such as the sequence of SEQ ID NO: 7 or other such sequences that are substantially identical to the sequences present in the primary transcript of the endogenous gene will be repressed. The introduced sequence is typically substantially identical to the endogenous gene targeted for suppression. Such identities are typically greater than about 50%, but higher identities (e.g., 80% or even 95%) are preferred because they result in more effective inhibition. The effect of co-suppression can also be applied to other proteins in similar families of genes that show homology or substantial homology. Fragments of a gene from one plant can be used directly, for example, to inhibit expression of homologous genes in different plant species.

In sense suppression, the introduced sequence, which requires less absolute identity, need not be full length relative to the primary transcript or fully processed mRNA. A higher degree of sequence identity in sequences shorter than the full length complements longer sequences of lesser identity. Furthermore, the introduced sequences need not have the same intron or exon pattern, and the identity of the non-coding segments may be equally effective. Sequences of at least 50 base pairs are preferred, with longer lengths of the introduced sequence being more preferred (see, e.g., the methods described in Jorgensen et al, U.S. Pat. No. 5,034,323).

Antisense inhibition

In antisense technology, nucleic acid fragments of genes from a desired plant are cloned, such as in SEQ ID NOS: 1, 3, 5, 6, or 7, and operably linked to an expression control region to synthesize an antisense strand of RNA. Next, the construct is transformed into a plant and the antisense strand of RNA is produced. In plant cells, antisense RNA has been shown to inhibit gene expression.

The nucleic acid fragment introduced in antisense suppression is typically substantially identical to at least a portion of the endogenous gene or genes being suppressed, but need not be identical. The nucleic acid sequences of the tobacco nicotine demethylase disclosed herein may be included in vectors designed to allow for the application of inhibition to other proteins in a family of genes exhibiting homology or substantial homology to the gene of interest. Fragments of a gene from one plant can be used directly, for example, to inhibit expression of homologous genes in different nicotiana species.

The introduced sequence need not be full length relative to the primary transcript or fully processed mRNA. In general, higher homology can be used to compensate for the use of shorter sequences. Moreover, the sequences introduced need not have identical intron or exon patterns, and homology of non-coding segments will be equally effective. Typically, such antisense sequences will generally be at least 15 base pairs in length, preferably about 15-200 base pairs, and more preferably 200-2,000 base pairs or longer. The antisense sequence may be complementary to all or part of the Gene to be inhibited (e.g., the tobacco nicotine demethylase promoter (SEQ ID NO: 6), exon, intron (SEQ ID NO: 5), or UTR (SEQ ID NO: 7), and as understood by those skilled in the art, the particular site or sites to which the antisense sequence binds and the length of the antisense sequence will vary depending on the degree of inhibition desired and the unique nature of the antisense sequence the transcription construct expressing the plant negative regulator antisense nucleotide sequence comprises, in the direction of transcription, a promoter, a sequence encoding an antisense RNA on the sense strand, and a transcription termination region, antisense sequences may be constructed and used as described, for example, in van der Krol et al. (Gene 72: 45-50, 1988); Rodermel et al. (Cell 55: 673-, 1988), 1990) (ii) a Weigel and Nilsson (Nature 377: 495-; cheung et al, (Cell 82: 383-; and Shewmaker et al (U.S. Pat. No. 5,107,065).

Dominant negative regulation

The transgenic plant can be assayed in an artificial environment or in the field to confirm that the transgene confers down-regulation of tobacco nicotine demethylase in the transgenic plant, which expresses a transgene encoding a dominant negative gene product of tobacco nicotine demethylase. Dominant negative transgenes were constructed according to methods known in the art. Typically, the dominant negative gene encodes a mutant negative regulator polypeptide of a tobacco nicotine demethylase that, when overexpressed, interferes with the activity of the wild-type enzyme.

Mutants

Plants having reduced expression or enzymatic activity of a tobacco nicotine demethylase can also be produced using standard mutagenesis methods. Methods for such mutagenesis include, but are not limited to, treatment of The seed with ethylmethylsulfate (Hildering and Verkerk, In, The use of induced mutations In plantarbrancing. Pergamon press, pp 317-. Types of mutations that may be present in the tobacco nicotine demethylase gene include, for example, point mutations, deletions, insertions, duplications and inversions. These mutations are desirably present in the coding region of the tobacco nicotine demethylase gene; however, mutations in the promoter region, and introns, or untranslated regions of the tobacco nicotine demethylase gene may also be desirable.

For example, a T-DNA insertion mutation can be used to generate an insertion mutation in a tobacco nicotine demethylase gene to down-regulate the expression of that gene. In theory, about 100,000 independent T-DNA inserts are required for a 95% probability of obtaining an insert in any given gene (McKinnet, Plant J.8: 613-. Polymerase Chain Reaction (PCR) analysis can be used to screen plants for T-DNA labeled lines. For example, a primer for one end of T-DNA may be designed, and another primer for a target gene may be designed, both of which may be used in a PCR analysis. If no PCR product is obtained, there is no insert in the target gene. In contrast, if a PCR product is obtained, there is an insert in the target gene.

Expression of the mutated tobacco nicotine demethylase may be assessed according to standard methods (e.g., those described herein), and optionally may be compared to expression of the unmutated enzyme. Mutant plants having reduced expression of a gene encoding a tobacco nicotine demethylase are desirable embodiments of the present invention when compared to unmutated plants.

Plant promoters

Examples of useful plant promoters according to the present invention are those having the sequence of SEQ ID NO: 6, or a fragment thereof that drives transcription. Another desirable promoter is a cauliflower mosaic virus promoter, such as the cauliflower mosaic virus (CaMV) promoter or the cassava vein mosaic virus (CsVMV) promoter. These promoters confer high levels of expression in most plant tissues, and the activity of these promoters is independent of the virally encoded protein. CaMV is the source of both the 35S and 19S promoters. Examples of plant expression constructs using these promoters are known in the art. In most tissues of transgenic plants, the CaMV 35S promoter is a strong promoter. The CaMV promoter is also highly active in monocots. Moreover, the activity of this promoter can be further increased (i.e., between 2-10 fold) by replication of the CaMV 35S promoter.

Other useful plant promoters include, but are not limited to, the nopaline synthase (NOS) promoter, the octopine synthase promoter, the figwort (figwort) mosaic virus (FMV) promoter, the rice actin promoter, and the ubiquitin promoter system.

Exemplary monocot plant promoters include, but are not limited to, commelina yellow mottle virus (commelinavirus) promoter, sugarcane badna virus promoter, rice tungro baculovirus promoter, maize streak virus element, and wheat dwarf virus promoter.

For certain applications, it may be desirable to produce a tobacco nicotine demethylase, such as a dominant negative mutant tobacco nicotine demethylase, at a suitable level, or at a suitable developmental time, in a suitable tissue. For this purpose, there are various classes of gene promoters, each with its own unique trait embodied in its regulatory sequence, that appear to be regulated in response to inducible signals such as environmental, hormonal and/or developmental information. These include, but are not limited to, gene promoters responsible for thermoregulatory gene expression, photoregulated gene expression (e.g., pea rbcS-3A; corn rbcS promoter; chlorophyll a/b binding protein gene found in pea; or Arassu promoter), hormone-regulated gene expression (e.g., abscisic acid (ABA) responsive sequences from wheat Em gene; ABA-induced HVA1 and HVA22, and rd29A promoter of barley and Arabidopsis (Arabidopsis)), and wound-induced gene expression (e.g., of Wuni), organ-specific gene expression (e.g., organ-specific gene expression of tuber-specific storage protein genes; organ-specific gene expression of 23-kDa zein gene from said corn; or organ-specific gene expression of French bean beta-phaseolin gene), or pathogen-induced promoters (e.g., PR-1, prp-1, or β -1, 3-glucanase promoters, a fungal-induced wrla promoter of wheat, and a nematode-induced promoter, TobRB7-5A and Hmg-1) of tobacco and celery, respectively).

Plant expression vectors

Typically, plant expression vectors include (1) a plant gene (e.g., a tobacco nicotine demethylase gene) cloned under the transcriptional control of 5 ' and 3 ' regulatory sequences (e.g., a tobacco nicotine demethylase promoter (SEQ ID NO: 6) and a 3 ' UTR region (SEQ ID NO: 7)) and (2) a dominant selectable marker. If desired, such plant expression vectors can also comprise, promoter regulatory regions (e.g., promoter regulatory regions conferring inducible or constitutive, pathogen or wound-induced, environmental or developmental regulation, or cell or tissue specific expression), transcription initiation start sites, ribosome binding sites, RNA processing signals, transcription termination sites, and/or polyadenylation signals.

Plant expression vectors may also optionally contain RNA processing signals, such as introns, which have been shown to be important for efficient RNA synthesis and accumulation. The positioning of RNA splice sequences can greatly affect the level of transgene expression in plants. In view of this fact, introns may be located upstream or downstream of the tobacco nicotine demethylase coding sequence in the transgene to alter the level of gene expression.

In addition to the 5 'regulatory control sequences described above, the expression vector may also comprise regulatory control regions, which are typically present in the 3' region of plant genes. For example, a 3' terminator region (e.g., the sequence of SEQ ID NO: 7) can be included in the expression vector to increase the stability of the mRNA. One such terminator region may be from the PI-II terminator region of potato. In addition, other commonly used terminators are derived from the octopine or nopaline synthase signal.

Plant expression vectors also typically contain dominant selectable marker genes that are used to identify those cells that have been transformed. Useful selectable genes for plant systems include the aminoglycoside phosphotransferase gene of transposon Tn5(Aph II), genes encoding antibiotic resistance genes, for example those encoding resistance to hygromycin, kanamycin, bleomycin, neomycin G418, streptomycin or spectinomycin. Genes required for photosynthesis can also be used as selectable markers for photosynthetic-deficient strains. Finally, genes encoding herbicide resistance can be used as selectable markers; useful herbicide resistance genes include those encoding the enzyme phosphinothricin acetyltransferase and conferring tolerance to the broad spectrum herbicide Basta(Bayer Cropscience Deutschland GmbH, Langenfeld, Germany). Other selectable markers include genes that provide resistance to other such herbicides such as glyphosate and the like, and imidazolinones, sulfonylureas, triazolopyrimidine herbicides such as chlorosulfuron, bromoxynil, dalapon and the like. In addition, the gene encoding dihydrofolate reductase can be used in combination with a molecule such as methatrexate.

The effective use of selectable markers is facilitated by determining the sensitivity of the plant cells to a particular selectable agent and determining the concentration of that agent that effectively kills most, if not all, of the transformed cells. Some useful antibiotic concentrations for tobacco transformation include, for example, 20-100. mu.g/ml (kanamycin), 20-50. mu.g/ml (hygromycin), or 5-10. mu.g/ml (bleomycin). Useful strategies for selecting transformants for herbicide resistance are described, for example, by Vasil (CellCulture and social Cell Genetics of Plants, Vol I, II, III laboratory procedures and the same Applications Academic Press, New York, 1984).

In addition to selectable markers, it may be desirable to use reporter genes. In some cases, a reporter gene may be used without a selectable marker. A reporter gene is a gene that is typically not present or expressed in a recipient organism or tissue. Reporter genes typically encode proteins that provide some phenotypic change or enzymatic property. Examples of such genes are provided in Weising et al (Ann. Rev. genetics 22: 421, 1988), which is incorporated herein by reference. Preferred reporter genes include, but are not limited to, the Glucuronidase (GUS) gene and the GFP gene.

After the plant expression vector is constructed, the vector can be introduced into a plant host using some standard method, thereby producing a transgenic plant. These methods include (1) Agrobacterium-mediated transformation (A.tumefaciens or A.rhizogenes) (see, e.g., Lichtenstein and Fuller In: Genetic Engineering, vol 6, PWJ Rigby, ed, London, Academic Press, 1987; and Lichtenstein, C.P., and Draper, J.,. In: DNA Cloning, VolII, D.M.Glover, ed, Oxford, IRI Press, 1985; U.S. Pat. Nos. 4,693,976, 4,762,785, 4,940,838, 5,004,863, 5,104,310, 5,149,645, 5,159,135, 5,177,624, 5,231,010, 5,463,174,174, 5,469,976, and 5,06763; and WO 3,0517626,645, 5,3976, 9,029,029,976; see, WO 3976, WO 3; see, WO patent application No. 4,02599,023976, WO 3; WO 3, WO patent application No. 4,02599,025932; see, WO 3, WO 7145,025932; see, WO 73, WO 3, (7) the vortexing method, or (8) the so-called whiskers method (see, e.g., Coffee et al, U.S. Pat. nos. 5,302,523 and 5,464,765). Types of plant tissues that can be transformed with the expression vector include embryonic tissues, type I and type II callus tissues, hypocotyls, meristematic tissues, and the like.

Once introduced into plant tissue, expression of the structural gene can be determined by any means known in the art, and expression can be measured as the amount of transcribed mRNA, synthesized protein, or gene silencing, as determined by metabolite monitoring by chemical analysis of secondary alkaloids in tobacco (as described herein; see also U.S. Pat. No. 5,583,021, incorporated herein by reference). Techniques for in vitro culture of plant tissues are known, and in many cases, techniques for regeneration into whole plants are known (see, e.g., U.S. patent nos. 5,595,733 and 5,766,900). Methods for delivering the introduced expression complexes into commercial cultivars are known to those skilled in the art.

Once plant cells expressing desired levels of nicotine demethylase (or nornicotine or NNN or both) are obtained, plant tissues and whole plants can be regenerated therefrom using methods and techniques well known in the art. The regenerated plants are then propagated by conventional means and the introduced genes can be delivered to other lines and cultivars by conventional plant breeding techniques.

The transgenic tobacco plant may bind nucleic acid of any part of a genomic gene in different orientations, either for down-regulation, e.g., in antisense orientation or in some form to induce RNAi, or for over-expression, e.g., in sense orientation. For increased expression of nicotine demethylase in nicotiana lines, overexpression of a nucleic acid sequence encoding a complete or functional portion of the amino acid sequence of a full-length tobacco nicotine demethylase gene is desirable.

Determination of the transcriptional or translational level of tobacco nicotine demethylase

Expression of tobacco nicotine demethylase can be measured, for example, by standard northern blot analysis using tobacco nicotine demethylase (or cDNA fragments) as a hybridization probe (Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, (2001), and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989)). Determination of RNA expression levels can also be assisted by reverse transcription PCR (rtPCR) including quantitative rtPCR (see, e.g., Kawasaki et al, in PCR Technology: Principles and Applications of DNA Amplification (H.A. Erlich, Ed.) Stockton Press (1989); Wang et al PCR Protocols: A Guide to Methods and Applications (M.A. Innis, et al, Eds.) Academic Press (1990); and Freeman et al, Biotechnology 26: 112-. Additional well-known techniques for determining expression of a tobacco nicotine demethylase gene include in situ hybridization, and fluorescent in situ hybridization (see, e.g., Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, (2001)). The standard techniques described above can also be used to compare expression levels between plants, for example, between plants having a mutation in the tobacco nicotine demethylase gene and control plants.

If desired, expression of the tobacco nicotine demethylase gene can be measured at the level of production of tobacco nicotine demethylase protein using the same general methods and standard protein analysis techniques including Bradford assay, spectrophotometry, and immunodetection techniques such as Western blotting or immunoprecipitation with tobacco nicotine demethylase-specific antibodies (Autobel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, (2001), and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold spring Harbor Laboratory, N.Y. (1989)).

Identification of tobacco nicotine demethylase modulators

Isolation of tobacco nicotine demethylase cDNA also facilitates identification of molecules that increase or decrease expression of tobacco nicotine demethylase. According to one method, the candidate molecule is added to the culture medium of cells expressing tobacco nicotine demethylase mRNA (e.g., prokaryotic cells such as e.coli or eukaryotic cells such as yeast, mammalian, insect or plant cells) at various concentrations. Next, the expression of tobacco nicotine demethylase is measured in the presence and absence of the candidate molecule using standard methods such as those mentioned herein.

Candidate modulators may be purified (or substantially pure) molecules or may be a component of a mixture of compounds. In a mixed compound assay, expression of tobacco nicotine demethylase is tested against a smaller and smaller subset of the candidate compound library (e.g., generated by standard purification techniques, such as HPLC) until a single compound or minimal mixture of compounds is demonstrated to alter expression of the tobacco nicotine demethylase gene. Molecules that promote a reduction in the expression of tobacco nicotine demethylase are believed to be particularly useful in the present invention. Modulators found to be effective at the level of expression or activity of nicotine demethylase from tobacco are considered to be effective in plants.

For agricultural applications, the molecules, compounds or agents identified using the methods disclosed herein may be used as chemicals that are used as sprays or dusts on the leaves of plants. The molecule, compound or agent may also be applied to a plant in combination with another molecule that provides a benefit to the plant.

Use of nicotine demethylase Gene promoters and untranslated regions

The promoter region of the nicotine demethylase genes described herein are ethylene inducible or are associated with plant senescence. Thus, the promoter may be used to drive the expression of any desired gene product to improve crop quality or to increase a particular trait. Because the tobacco nicotine demethylase promoter (e.g., SEQ ID NO: 6) is inducible and expressed at specific stages of the plant life cycle, constructs comprising the promoter can be introduced into plants to express unique genes involved in the biosynthesis of aroma and aromatic products by secondary metabolites. Examples of such compounds are compounds in the terpenoid pathway, other alkaloids, plant hormones, flavonoids or sugar-containing fractions. The tobacco nicotine demethylase promoter may also be used to increase or alter the expression of structural sugars or proteins, which affect the end use properties. In addition, the tobacco nicotine demethylase promoter may also be combined with heterologous genes, including genes involved in the biosynthesis of nutritional products, pharmaceutical agents, or industrial substances. The promoter may be used to drive down-regulation of genes endogenous to tobacco, including nicotine demethylase or other genes involved in alkaloid biosynthesis and or in other pathways.

Moreover, the promoter region of the tobacco nicotine demethylase gene (e.g., SEQ ID NO: 6) or the 3' UTR of the tobacco nicotine demethylase gene (e.g., SEQ ID NO: 7) may also be used in any site-directed gene silencing approach such as T-DNA tagging, gene capture, and homologous recombination to alter the expression pattern of a target gene, as described herein. Promoter motifs, which can be readily expressed in promoter sequences, such as SEQ ID NOs: 6. Ideally, the tobacco nicotine demethylase promoter region or other transcriptional regulatory region is used to alter chemical properties such as nornicotine content and nitrosamine levels in plants.

In addition, any portion of the tobacco nicotine demethylase gene may be used as a genetic marker to isolate the relevant gene, promoter or regulatory region, or to screen for demethylase genes in other tobacco or nicotine species.

Product of

Tobacco products having reduced nitrosamine content are prepared according to standard methods known in the art using any of the tobacco plant materials described herein. In one embodiment, a tobacco product is prepared using plant material obtained from genetically modified processed tobacco having less than about 5mg/g, 4.5mg/g, 4.0mg/g, 3.5mg/g, 3.0mg/g, 2.5mg/g, 2.0mg/g, 1.5mg/g, 1.0mg/g, 750 μ g/g, 500 μ g/g, 250 μ g/g, 100 μ g/g, 75 μ g/g, 50 μ g/g, 25 μ g/g, 10 μ g/g, 7.0 μ g/g, 5.0 μ g/g, 4.0 μ g/g, 2.0 μ g/g, 1.0 μ g/g, 0.5 μ g/g, 0.4 μ g/g, 0.2 μ g/g, 0.1 μ g/g, 0.05 μ g/g, or a reduced amount of nornicotine or NNN of 0.01 μ g/g, or wherein the percentage of secondary alkaloids relative to the total alkaloid content comprised therein is less than 90%, 70%, 50%, 30%, 10%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, or 0.1%. That is, the processed tobacco is prepared from a genetically modified tobacco plant. The phrase "reduced amount" is intended to mean that less amount of nornicotine or NNN, or both, is present in a transgenic tobacco plant, tobacco, or tobacco product from the same species treated in the same manner, which has not been made into transgenic material having reduced nornicotine or NNN, as compared to that found in a naturally occurring tobacco plant, tobacco, or tobacco product from the same species treated in the same manner. Thus, in certain instances, the same species of naturally occurring tobacco processed in the same manner is used as a control by which to measure whether a reduction in nornicotine or NNN has been obtained by the methods described herein. The levels of nornicotine and NNN were measured according to methods well known in the tobacco art.

The following examples illustrate the method of practicing the invention and should be understood as illustrative and not limiting of the scope of the invention, which is defined in the appended claims.

Example 1

Development and ethylene treatment of plant tissues

Growth of plants

Plants were grown in pots and in a greenhouse for 4 weeks. Seedlings at 4 weeks of age were transplanted into individual pots and grown in the greenhouse for 2 months. During the growth process the plants were watered 2 times a day with water containing 150ppm NPK fertilizer. The developed green leaves were separated from the plants and subjected to the following ethylene treatment.

Cell line 78379

Tobacco line 78379, which is a burley tobacco line given by kentucky university, was used as a source of plant material. One hundred plants were grown according to the standard for growing tobacco, transplanted and marked with different numbers (1-100). Fertilization and field management were performed as recommended.

Three quarters of the 100 plants converted between 20 and 100% of the nicotine to nornicotine. Less than 5% of the nicotine is converted to nornicotine in one quarter of the 100 plants. Plant No. 87 had the least transformation (2%) and plant No. 21 had 100% transformation. Plants transformed by less than 3% were classified as non-transformants. Self-pollinated seeds of plant No. 87 and plant No. 21 were prepared, as well as crossed (21x87 and 87x21) seeds to study genetic and phenotypic differences. Plants from selfed 21 were transformants, while 99% of the selfed lines from 87 were non-transformants. The other 1% of plants from 87 showed low transformation (5-15%). The plants from the backcross were all transformants.

Cell line 4407

Nicotiana line 4407 is used as a source of plant material, said tobacco line 4407 being a burley line. Consistent and representative plants (100) were selected and marked. 97 out of 100 plants were non-transformants and three were transformants. Plant number 56 had the least amount of transformation (1.2%) and plant number 58 had the highest level of transformation (96%). Both plants were used to prepare self-pollinated seeds and hybrid seeds.

Plants from selfed 58 were isolated at a 3: 1 ratio of transformants to non-transformants. Plants 58-33 and 58-25 were identified as homozygous transformant and non-transformant plant lines, respectively. The stable transformation of 58-33 was confirmed by analysis of its progeny.

Cell line PBLB01

PBLB01 is a burley tobacco line developed by ProfiGen, inc and used as a source of plant material. The transformant plants were selected from the starting seeds of PBLB 01.

Ethylene treatment process

Green leaves were removed from greenhouse-cultivated plants for 2-3 months and sprayed with 0.3% ethylene solution (Prep brandEthephon (Rhone-Poulenc)). Each sprayed leaf was hung on a treatment rack equipped with a humidifier and coated with plastic. During the treatment, the sample leaves were periodically sprayed with an ethylene solution. Approximately 24-48 hours after ethylene treatment, the leaves were harvested for RNA extraction. Another aliquot was taken for metabolic component analysis to determine the concentration of leaf metabolites and more specific target components such as various alkaloids.

As an example, the alkaloid analysis may be performed as follows. Samples (0.1g) were shaken at 150rpm with 0.5ml 2N NaOH and 5ml extraction solution containing quinoline and methyl tert-butyl ether as internal standards. Samples were analyzed on HP 6890 GC equipped with FID detector. A temperature of 250 ℃ was used for the detector and syringe. An HP column (30m-0.32nm-1mm) composed of fused silica cross-linked with 5% phenol and 95% polymethylsiloxane (methyl silicone) was used at a temperature gradient of 110 ℃ and 185 ℃ of 10 ℃ per minute. At 100 ℃ by 1.7cm³min^-1At a cracking ratio of 40: 1, the column was operated with helium as carrier gas at an injection volume of 2: 1.

Example 2

RNA isolation

For RNA extraction, middle leaves from two months old greenhouse grown plants were treated with ethylene as described above. Samples from 0 and 24-48 hours were used for RNA extraction. In some cases, leaf samples in the senescence process were taken from plants 10 days after the removal of flower heads. These samples were also used for extraction. Rneasy Plant Mini Kit was utilized according to the manufacturer's protocol(Qiagen, Inc., Valencia, California) Total RNA was isolated.

The tissue samples were ground to a fine powder under liquid nitrogen using a DEPC treated mortar and pestle. Approximately 100mg of ground tissue was transferred into a sterile 1.5ml Eppendorf tube. This sample tube was placed in liquid nitrogen until all samples were collected. Subsequently, 450 μ l of buffer RLT (added mercaptoethanol) as provided in the kit was added to each individual tube. The sample was vortexed vigorously and incubated at 56 ℃ for 3 minutes. The lysate was then applied to a QIAshreder placed in a 2ml collection tubeSpin on column and centrifuge at maximum speed for 2 minutes. The flow-through was collected and 0.5 volume of ethanol was added to the cleared lysate. Mix the sample well and transfer to Rneasy in a 2ml collection tubeOn a miniature centrifugal column. The samples were centrifuged at 10,000rpm for 1 minute. Next, 700. mu.l of buffer RW1 was pipetted into RneasyColumn and centrifuge at 10,000rpm for 1 minute. Buffer RPE pipetted into Rneasy in a fresh collection tubeColumn and centrifuge at 10,000rpm for 1 minute. Buffer RPE was again added to RneasySpin column and centrifuge at maximum speed for 2 minutes to dry the membrane. To remove any residual ethanol, the membranes were placed in a separate collection tube and centrifuged again at maximum speed for 1 minute. RneasyThe column was transferred into a new 1.5ml collection tube and 40. mu.l of RNase-free water was pipetted directly into RneasyOn the membrane. This final elution tube was centrifuged at 10,000rpm for 1 minute. The quality and quantity of total RNA were analyzed by denaturing formaldehyde gel and spectrophotometer.

Oligotex was used following the manufacturer's protocolPoly (a) RNA was isolated using a poly a + RNA purification kit (Qiagen Inc.). Approximately 200. mu.g total RNA in a maximum volume of 250. mu.l was used. Buffer OBB was added in a volume of 250. mu.l and Oligotex was added in a volume of 15. mu.lThe suspension was added to 250. mu.l of total RNA. The components were mixed well by pipetting and incubated at 70 ℃ for 3 minutes on a heating module. The sample was then left at room temperature for approximately 20 minutes. Oligotex was centrifuged at maximum speed for 2min: the mRNA complex precipitates. All material except 50. mu.l of supernatant was removed from the microcentrifuge tube. The sample was further treated with OBB buffer. Oligotex was vortexed: the mRNA pellet was resuspended in 400. mu.l of buffer OW 2. This mixture was transferred to a small spin column placed in a new tube and centrifuged at maximum speed for 1 minute. The column was transferred to a new tube and an additional 400. mu.l of buffer OW2 was added to the column. The tube was then centrifuged at maximum speed for 1 minute. The spin column was transferred to a final 1.5ml microcentrifuge tube. The sample was eluted with 60. mu.l of hot (70 ℃) buffer OEB. The poly a products were analyzed by denaturing formaldehyde gel and spectrophotometric analysis.

Example 3

Reverse transcription PCR

First strand cDNA was prepared using SuperScript reverse transcriptase (Invitrogen, Carlsbad, California) according to the manufacturer's protocol. The RNA/oligo dT primer mix enriched in poly A + consists of less than 5. mu.g total RNA, 1. mu.l of 10mM dNTP mix, 1. mu.l oligo d (T)_12-18(0.5. mu.g/. mu.l), and up to 10. mu.l of DEPC treated water. Each sample was incubated at 65 ℃ for 5 minutes and then placed on ice for at least 1 minute. The reaction mixture was prepared by adding each of the following components in sequence: 2 μ l10X RT buffer, 4 μ l 25mM MgCl₂2. mu.l of 0.1M DTT, and 1. mu.l of RNase OUT recombinant RNase inhibitor. An additional 9. mu.l of reaction mixture was pipetted into each RNA/primer mixture and gently mixed. This was incubated at 42 ℃ for 2 minutes and 1. mu.l of Super Script II RT was added to each tube. The tubes were incubated at 42 ℃ for 50 minutes. The reaction was stopped at 70 ℃ for 15 minutes and cooled on ice. Samples were collected by centrifugation and 1. mu.l of RNase H was added to each tube and incubated at 37 ℃ for 20 minutes. A second PCR was performed with 200pmoles of forward primer and 100pmoles of reverse primer (a mixture of 18nt oligo d (T) with 1 random base after).

The reaction conditions were 94 ℃ for 2 minutes and then 40 PCR cycles of 94 ℃ for 1 minute, 45 ℃ to 60 ℃ for 2 minutes, and 72 ℃ for 3 minutes, with an additional extension at 72 ℃ for 10 min. 10 microliter of the amplified sample was analyzed by electrophoresis using a 1% agarose gel. The correct size fragment was purified from agarose gel.

Example 4

Generation of PCR fragment groups

The PCR fragment from example 3 was ligated into the pGEM-T Easy vector (Promega, Madison, Wisconsin) according to the manufacturer's instructions. The ligation products were transformed into JM109 competent cells and plated on LB medium plates for blue/white selection. Colonies were selected and grown overnight at 37 ℃ in 96-well plates with 1.2ml LB medium. Frozen stock solutions were prepared for all selected colonies. Plasmid DNA was purified from the plates using a Wizard SV Miniprep kit (Promega) using a Beckman's Biomeck 2000 Miniprep robot. Plasmid DNA was eluted with 100. mu.l water and stored in 96-well plates. The plasmid was digested by EcoRl and analyzed using 1% agarose gel to determine the amount of DNA and the size of the insert. The plasmid containing the 400-and 600-bp insert was sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, Calif.). Sequences were aligned to the GenBank database by BLAST search. P450 related fragments were identified and further analyzed. Alternatively, p450 fragments are isolated from a subtractive library. These fragments were also analyzed as described above.

Example 5

cDNA library construction

A cDNA library was constructed by preparing total RNA from ethylene treated leaves as follows. First, total RNA was extracted from leaves of the ethylene-treated tobacco line 58-33 using a modified acid phenol and chloroform extraction method. The method was modified to use one gram of tissue that was ground and then vortexed in 5ml of extraction buffer (100mM Tris-HCl, pH 8.5; 200mM NaCl; 10mM EDTA; 0.5% SDS) to which 5ml of phenol (pH5.5) and 5ml of chloroform were added. The extracted sample was centrifuged and the supernatant was retained. This extraction step was repeated 2-3 times until the supernatant appeared clear. About 5ml of chloroform was added to remove traces of phenol. RNA was precipitated from the combined supernatant fractions by adding 3 volumes of ethanol and 1/10 volumes of 3M NaOAc (pH5.2) and stored at-20 ℃ for 1 hour. After transfer into a Corex glass container, the RNA fraction was centrifuged at 9,000RPM for 45 minutes at 4 ℃. The precipitate was washed with 70% ethanol and centrifuged at 9,000RPM for 5 minutes at 4 ℃. After drying the precipitate, the precipitated RNA was dissolved in 0.5ml RNase-free water. The quality and quantity of total RNA were analyzed by denatured formaldehyde gel and spectrophotometer, respectively.

The resulting total RNA was used to isolate poly A + RNA using oligo (dT) cellulose method (Invitrogen) and microcentrifuge column (Invitrogen) by the following protocol. Approximately 20mg of total RNA was purified twice to obtain high quality poly A + RNA. The poly A + RNA products were analyzed by performing denaturing formaldehyde gels followed by RT-PCR of known full-length genes to ensure high quality mRNA.

Next, a cDNA library was generated using poly A + RNA as a template using a cDNA synthesis kit, a ZAP-cDNA synthesis kit, and a ZAP-cDNA Gigapack IIIgold cloning kit (Stratagene, La Jolla, California). The method includes following a specified manufacturer's protocol. Approximately 8. mu.g of poly A + RNA was used to construct the cDNA library. Analysis of the primary library revealed approximately 2.5x10⁶-1x10⁷pfu. Library quality background testing was done by complementation assay using IPTG and X-gal, where the recombinant plaques were expressed more than 100-fold above the background reaction.

More quantitative library analysis by random PCR showed that the average size of the inserted cDNA was about 1.2 kb. The method uses a two-step PCR method. For the first step, reverse primers were designed based on preliminary sequence information obtained from the p450 fragment. The corresponding gene was amplified from the cDNA library using the designed reverse primer and T3 (forward) primer. The PCR reactions were subjected to agarose electrophoresis and the corresponding high molecular weight bands were excised, purified, cloned and sequenced. In the second step, a new primer designed as a forward primer from the 5 'UTR or the starting coding region of p450 was used in subsequent PCR together with a reverse primer (designed from the 3' UTR of p450) to obtain a full-length p450 clone.

With the exception of the reverse primer, a p450 fragment was generated from the constructed cDNA library by PCR amplification as described in example 3. The T7 primer located downstream of the cDNA insert plasmid was used as the reverse primer. The PCR fragments were isolated, cloned and sequenced as described in example 4.

The full-length p450 gene was isolated from the constructed cDNA library by this PCR method. The full-length gene was cloned using gene-specific reverse primers (designed from the sequence downstream of the p450 fragment) and forward primers (T3 on the library plasmid). The PCR fragments were isolated, cloned and sequenced. If necessary, a second PCR step is applied. In a second step, a new forward primer designed from the 5 'UTR of cloned p450s was used in a subsequent PCR reaction with a reverse primer designed from the 3' UTR of p450 clone to obtain a full-length p450 clone. Clones were subsequently sequenced.

Example 6

Characterization of cloned fragments-reverse southern blot analysis

A non-radioactive large scale reverse southern blot assay was performed on all p450 clones identified in the above examples to detect differential expression. Very different expression levels were observed between different p450 clusters. Further real-time detection was performed for those with high expression.

Non-radioactive southern blotting procedures were performed as follows.

1) Total RNA was extracted from leaves of ethylene-treated and untreated transformants (58-33) and non-transformants (58-25) using the Qiagen Rnaesasy kit as described in example 2.

2) The probe was generated by labeling the biotin tail with single-stranded cDNA derived from poly a + rich RNA generated in the above step. This labelled single stranded cDNA was generated by RT-PCR of transformant and non-transformant total rna (invitrogen) as described in example 3, except that biotinylated oligo dT was used as primer (Promega). These were used as probes for hybridization with cloned DNA.

3) Plasmid DNA was digested with the restriction enzyme EcoR1 and electrophoresed on an agarose gel. The gel was simultaneously dried and transferred to two nylon membranes (Biodyne B). One membrane was hybridized with the transformant probe and the other with a non-transformant probe. Membranes were UV-crosslinked prior to hybridization (auto-crosslinking device, 254nm, Stratagene, Stratalinker).

Alternatively, the insert was PCR amplified from each plasmid using the sequences T3 and SP6 located on both arms of the p-GEM plasmid as primers. PCR products were analyzed by electrophoresis on 96-well pre-run (Ready-to-run) agarose gels. The confirmed inserts were spotted on two nylon membranes. One membrane was hybridized with the transformant probe and the other with a non-transformant probe.

4) Washing stringency was modified, membranes were hybridized and washed according to the manufacturer's instructions (zone MaxSence kit, zone Diagnostics, Inc, Farmingdale, NY). The membrane was prehybridized at 42 ℃ for 30min with hybridization buffer (2 XSSC buffered formamide containing detergent and hybridization enhancer) and hybridized with 10. mu.l of denatured probe overnight at 42 ℃. The membrane was then washed 1 time for 10min at room temperature in 1X hybridization wash buffer and 4 times for 15min at 68 ℃. The membrane may be used in detection operations.

5) The washed membranes were tested by alkaline phosphatase labeling followed by NBT/BCIP colorimetric assay as described in the manufacturer's test methods (Enzo Diagnostics, Inc.). The membrane was blocked with 1 × blocking solution for 1 hour at room temperature, washed 3 times with 1 × detection reagent for 10min, washed 2 times with 1 × pre-color reaction buffer for 5min, and then the spots were developed in color solution for 30-45min until spots appeared. All reagents were supplied by the manufacturer (Enzodiagnostics, Inc). In addition, large scale reverse DNA assays were also performed using the KPL DNA hybridization and detection kit according to the manufacturer's instructions (KPL, Gaithersburg, Maryland).

Example 7

Characterization of clones-northern blot analysis

As an alternative to southern blot analysis, some membranes were hybridized and detected as described in the examples of northern blot assays. RNA hybridization was used to detect mRNA differentially expressed in nicotiana as follows.

Probes were prepared from the cloned p450 using a random priming method (Megaprime DNA labelling systems,amersham Biosciences). The following components were mixed: 25ng of denatured DNA template; 4 μ l of each unlabeled dTTP, dGTP and dCTP; 5. mu.l of reaction buffer; p³²Labeled dATP and 2 μ l Klenow I; and H₂O, to bring the reaction to 50. mu.l. The mixture was incubated at 37 ℃ for 1-4 hours and stopped with 2. mu.l of 0.5M EDTA. The probes were denatured by incubation at 95 ℃ for 5 minutes prior to use.

RNA samples were prepared from fresh leaves of ethylene-treated and untreated pairs of tobacco lines. In some cases, RNA rich in poly A + is used. With DEPC H₂O (5-10. mu.l) approximately 15. mu.g total RNA or 1.8. mu.g mRNA (RNA and mRNA extraction method as described in example 5) were brought to equivalent volumes. The same volume of loading buffer (1 XMOPS; 18.5% formaldehyde; 50% formamide; 4% Ficoll 400; bromophenol blue) and 0.5. mu.l EtBr (0.5. mu.g/. mu.l) were added. The sample is then denatured in preparation for RNA separation by electrophoresis.

Samples were electrophoresed on formaldehyde gel (1% agarose, 1XMOPS, 0.6M formaldehyde) with 1XMOP buffer (0.4M morpholinopropanesulfonic acid; 0.1M sodium acetate-3 xH 2O; 10mM EDTA; adjusted to pH 7.2 with NaOH). RNA was transferred to Hybond-N + membranes (Nylon, Amersham Pharmacia Biotech) by capillary method in 10 XSSC buffer (1.5 MNaCl; 0.15M sodium citrate) for 24 h. Membranes with RNA samples were UV cross-linked prior to hybridization (auto-cross-linker, 254nm, Stratagene, Stratalinker).

The membrane was prehybridized for 1-4 hours at 42 ℃ with 5-10ml of prehybridization buffer (5 XSSC; 50% formamide; 5 XDenhardt's-solution; 1% SDS; 100. mu.g/ml heat denatured sheared non-homologous DNA). The old pre-hybridization buffer was discarded and new pre-hybridization buffer and probe were added. Hybridization was carried out overnight at 42 ℃. The membrane was washed with 2x SSC at room temperature for 15 minutes, followed by 2x SSC.

Northern blot analysis was performed on nicotiana tissues obtained from both transformants and non-transformant burley lines, both induced by ethylene treatment, using full length clones. The objective was to identify full-length clones that showed increased expression in the ethylene-induced transformant line relative to the ethylene-induced non-transformant burley tobacco line. By doing so, the functional relationship of the full-length clones can be determined by comparing the biochemical differences in leaf composition between the transformant and non-transformant lines.

Example 8

Immunodetection of p450s encoded by cloned genes

Peptide regions corresponding to 20-22 amino acids long were selected from three p450 clones, which 1) had low or no homology to other clones and 2) had good hydrophilicity and antigenicity. The amino acid sequences of the peptide regions selected from the individual p450 clones are listed below. The synthetic peptide was conjugated to KHL and then injected into rabbits. Antisera were collected 2 and 4 weeks after the 4 th injection (Alpha Diagnostic int. inc. san Antonio, TX).

D234-AD1 DIDGSKSKLVKAHRKIDEILG(SEQ ID NO：8)

D90a-BB3 RDAFREKETFDENDVEELNY(SEQ ID NO：9)

D89-AB1 FKNNGDEDRHFSQKLGDLADKY(SEQ ID NO：10)

Antisera were examined by western blot analysis for cross-reactivity with target proteins from tobacco plant tissue. Crude protein extracts were obtained from the middle leaves of the ethylene-treated (0-40 hours) transformants and non-transformant strains. The protein concentration of the extract was determined using the RC DC protein assay kit (BIO-RAD) according to the manufacturer's protocol.

Two milligrams of protein were loaded onto each lane and the proteins were separated on a 10% -20% gradient gel using the Laemmli SDS-PAGE system. Proteins were transferred from the gel onto PROTRAN nitrocellulose transfer membrane (Schleicher & Schuell) using Trans-Blot Semi-Dry cells (BIO-RAD). The target p450 protein was detected and visualized using the ECL Advance Western Blotting detection kit (Amersham Biosciences). Primary antibodies against synthetic KLH conjugates were prepared in rabbits. Secondary antibodies against rabbit IgG conjugated to peroxidase were purchased from Sigma. Both primary and secondary antibodies were used at 1: 1000 dilution. The antibody showed strong reactivity to a single band on the western blot, suggesting that the antisera was monospecific for the target peptide of interest. Antisera were also cross-reactive with synthetic peptides conjugated to KLH.

Example 9

Nucleic acid identity and structural relatedness of isolated nucleic acid fragments

P450 fragments of more than 100 clones were sequenced in conjunction with northern blot analysis to determine their structural relationship. The method uses a forward primer based on either of two common p450 motifs located near the carboxy terminus of the p450 gene. The forward primer corresponds to the cytochrome p450 motif FXPERF (SEQ ID NO: 11) or GRRXCP (A/G) (SEQ ID NO: 12). The reverse primers used were standard primers from the plasmid, SP6 or T7 located on both arms of the pGEM plasmid, or standard primers from the poly A tail. The method used is described below.

The concentration of the starting double stranded DNA was evaluated spectrophotometrically according to the manufacturer's protocol (Beckman Coulter). The template was diluted with water to the appropriate concentration, denatured by heating at 95 ℃ for 2 minutes, and then placed on ice. Sequencing reactions were prepared on ice using 0.5-10. mu.l of denatured DNA template, 2. mu.l of 1.6pmol of forward primer, 8. mu.l of DTCS Quick Start Master Mix, and a total volume of 20. mu.l with water. The thermal cycling program consisted of 30 cycles of the following cycles: 96 ℃ for 20 seconds, 50 ℃ for 20 seconds, and 60 ℃ for 4 minutes, followed by holding at 4 ℃.

The sequencing reaction was stopped by adding 5. mu.l of stop buffer (equal volumes of 3M NaOAc and 100mM EDTA and 1. mu.l of 20mg/ml glycogen). The sample was precipitated with 60. mu.l of cold 95% ethanol and centrifuged at 6000Xg for 6 minutes. The ethanol was discarded. The precipitate was washed twice with 200. mu.l of cold 70% ethanol. After the precipitate was dried, 40. mu.l of SLS solution was added and the precipitate was resuspended. Covering with a layer of mineral oil. The samples were then placed on a CEQ 8000 autosequencer for further analysis.

To confirm the nucleic acid sequence, the nucleic acid sequence was re-sequenced in both directions using the forward primer or plasmid of the FXPERF (SEQ ID NO: 11) or GRRXCP (A/G) (SEQ ID NO: 12) region of the p450 gene or the reverse primer of the poly A tail. All sequencing was performed at least twice in both directions.

The nucleic acid sequences of the cytochrome p450 fragments were compared with each other from the coding region corresponding to the first nucleic acid after the motif region encoding GRRXCP (A/G) (SEQ ID NO: 12) up to the stop codon. This region was taken as an indication of genetic diversity between p450 proteins. Among the 70 excess genes, a large number of genetically distinct p450 genes were observed, similar to that of other plant species. After comparing nucleic acid sequences, it was found that genes can be inserted into different sequence groups based on their sequence identity. The best unique group of p450 members was found to be those sequences with 75% or greater nucleic acid identity. (see, for example, Table 1 in the publication of the US 2004/0162420 patent application, which is incorporated herein by reference). Reducing percent identity resulted in significantly larger groups. It was observed that the preferred group was those sequences having 81% or more nucleic acid identity, the more preferred group was those having 91% or more nucleic acid identity, and the most preferred group was those sequences having 99% or more nucleic acid identity. Most arrays contain at least two members and often three or more members. No other was repeatedly found, suggesting that the method used was able to isolate low and high expression mRNA in the tissues used.

The gene chip technology was used to identify genes differentially expressed in the transformant versus the non-transformant tobacco lines, and D121-AA8 was determined to have a reproducible induction in the ethylene-treated transformant line. Based on these results, the D121-AA8 gene (the cDNA sequence of which is the sequence of SEQ ID NO: 3; FIG. 3) was identified as a tobacco nicotine demethylase target gene.

In view of The p450 nomenclature, tobacco nicotine demethylase genes are novel and belong to The CYP82E class (The Arabidopsis Genome Initiative (AGI) and The Arabidopsis Information Resource (TAIR); Frank, Plant Physiol.110: 1035-; schopper and Ebel, mol.gen.genet.258: 315-322, 1998; and Takemoto et al, Plant Cell physiol.40: 1232-1242, 1999).

Example 10

Biochemical analysis of tobacco nicotine demethylase

Biochemical analysis, for example, as described in the previously filed application incorporated herein by reference, the amino acid sequence of SEQ id no: 3 encodes a tobacco nicotine demethylase (SEQ ID NO: 4; FIG. 3).

In particular, the candidate clone (D121-AA8) was determined to function as a gene encoding nicotine demethylase by assaying the enzymatic activity of p450 heterologously expressed in yeast cells as follows.

1. Construction of Yeast expression vectors

The putative protein coding sequence for the tobacco nicotine demethylase-encoding cDNA (D121-AA8) was cloned into the yeast expression vector pYeDP 60. Appropriate BamHI and MfeI sites (underlined below) were introduced upstream of the translation initiation codon (ATG) or downstream of the termination codon (TAA) by PCR primers containing these sequences. Mfel on the amplified PCR product was compatible with the EcoRI site on the vector. The primer used for amplifying cDNA was 5' -TAGCTACGCGGATCCATGCTTTCTCCCATAGAAGCC-3 '(SEQ ID NO: 27) and 5' -CTGGATCACAATTGTTAGTGATGGTGATGGTGATGCGATCCTCTATAAAGCTCAGGTGCCAGGC-3' (SEQ ID NO: 28). A sequence fragment encoding nine additional amino acids, including six histidines, at the C-terminus of the protein was incorporated into the reverse primer to facilitate expression of the post-induction 6-His tagged p 450. The PCR product was ligated into the pYeDP60 vector after enzymatic digestion in the sense orientation of the reference GAL10-CYC1 promoter. Correct construction of the yeast expression vector was confirmed by restriction enzyme analysis and DNA sequencing.

2. Yeast transformation

A WAT11 yeast strain modified to express the Arabidopsis NADPH-cytochrome p450 reductase ATR1 was transformed with the pYeDP60-p450cDNA plasmid. 50 microliters of WAT11 yeast cell suspension was mixed with-1. mu.g of plasmid DNA in a cuvette with an electrode gap of 0.2 cm. A2.0 kV pulse was applied to the Eppendorf electroporator (Model 2510). Cells were plated onto SGI plates (5g/L bactocasamino acids, 6.7g/L yeast nitrogen base without amino acids (nitrogenbase), 20g/L glucose, 40mg/L DL-tryptophan, 20g/L agar). Transformants were confirmed by PCR analysis performed directly on randomly selected colonies.

3. Expression of p450 in transformed yeast cells

A single yeast colony was used to inoculate 30mL of SGI medium (5g/L bactocaamino acids, 6.7g/L yeast nitrogen base without amino acids, 20g/L glucose, 40mg/L DL-tryptophan) and grown at 30 ℃ for approximately 24 hours. Aliquots of this culture were diluted 1: 50 into 1000mL YPGE medium (10g/L yeast extract, 20g/L bactopeptone, 5g/L glucose, 30mL/L ethanol) and cultured until glucose was completely depleted, as indicated by colorimetric changes on a Diastix urinalysis reagent strip (Bayer, Elkhart, IN). The induction of clone P450 was initiated by adding DL-galactose to a final concentration of 2%. Cultures were incubated for an additional 20 hours for in vivo activity assays or microsome preparations prior to use.

WAT11 yeast cells expressing pYeDP60-CYP71D20 (p 450 catalyzing hydroxylation of 5-epi-aristocine and 1-deoxyapdiol in tobacco) were used as controls for p450 expression and enzyme activity assays.

To evaluate the effectiveness of yeast expression of p450 in more detail, a simplified CO differential spectroscopy analysis was performed. The simplified CO spectrum shows a peak at 450nm for proteins from all four p450 transformed yeast strains. No similar peaks were observed in the microsomes of the control yeast or vector control yeast. The results show that the p450 protein is efficiently expressed in a yeast strain with pYeDP60-CYP 450. The concentration of p450 protein expressed in the yeast microsomes was from 45 to 68nmole/mg total protein.

4. In vivo enzyme assay

By feeding DL-nicotine (pyrrolidine-2-¹⁴C) To determine the activity of nicotine demethylase in transformed yeast cells. Will be provided with¹⁴C-labeled nicotine (54mCi/mmol) was added to 75. mu.l of galactose-induced culture to a final concentration of 55. mu.M. The assay cultures were incubated for 6 hours with shaking in 14ml polypropylene tubes and extracted with 900. mu.l of methanol. After centrifugation, 20 μ l of the methanol extract was separated by rp-HPLC and the nornicotine fraction was quantified by LSC.

The control culture of WAT11(pYeDP60-CYP71D20) did not convert nicotine to nornicotine, indicating that the WAT11 yeast strain did not contain endogenous enzyme activities capable of catalyzing the step of bioconversion of nicotine to nornicotine. In contrast, yeast expressing the tobacco nicotine demethylase gene produced detectable amounts of nornicotine, showing that SEQ ID NO: 3 nicotine demethylase activity of the translation product.

5. Yeast microsome preparation

After galactose induction for 20 hours, yeast cells were collected by centrifugation and washed twice with TES-M buffer (50mM Tris-HCl, pH 7.5, 1mM EDTA, 0.6M sorbitol, 10mM 2-mercaptoethanol). The pellet was resuspended in extraction buffer (50mM Tris-HCl, pH 7.5, 1mM EDTA, 0.6M sorbitol, 2mM 2-mercaptoethanol, 1% bovine serum albumin, 1 tablet/50 ml protease inhibitor cocktail (Roche)). The cells were then disrupted with glass beads (diameter 0.5mm, Sigma) and the cell extract was centrifuged at 20,000Xg for 20min to remove cell debris. The supernatant was ultracentrifuged at 100,000Xg for 60min, and the resulting pellet contained the microsomal fraction. The microsomal fraction was suspended at a protein concentration of 1mg/mL in TEG-M buffer (50mM Tris-HCl, pH 7.5, 1mM EDTA, 20% glycerol and 1.5mM 2-mercaptoethanol). Microsome preparations were stored in liquid nitrogen freezers for use.

6. Determination of enzyme Activity in Yeast microsome preparations

Determination of nicotine demethylase activity was performed using yeast microsome preparations. In particular, DL-nicotine (pyrrolidine-2-¹⁴C) Obtained from Moravek Biochemicals and having a specific activity of 54 mCi/mmol. Chlorpromazine (CPZ) and oxidized cytochrome C (cyt.c), both P450 inhibitors, purchased from Sigma. Reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is a cytochrome P450 binding site that is formed by NADPH: typical electron donors for cytochrome P450 reductases. No NADPH was present in the control incubations. Conventional enzyme assays include microsomal protein (about 1mg/ml), 6mM NADPH, and 55. mu.M¹⁴C-labeled nicotine. When used, concentrations of CPZ and Cyt.C were 1mM and 100. mu.M, respectively. The reaction was carried out at 25 ℃ for 1 hour and stopped by adding 300. mu.l of methanol to each 25. mu.l of the reaction mixture. After centrifugation, 20. mu.l of methanol extract was separated using an Inertsil ODS-33. mu.l (150X4.6mm) column from Varian in a reversed-phase High Performance Liquid Chromatography (HPLC) system (Agilent). The constant solvent (isocratic) mobile phase was a mixture of methanol and 50mM potassium phosphate buffer, pH 6.25, at a ratio of 60: 40(v/v) and a flow rate of 1 ml/min. The nornicotine peak was collected and quantified with a 2900tri-carb Liquid Scintillation Counter (LSC) (PerkinElmer), which was determined by comparison with authentic unlabeled nornicotine. After 1 hour incubation based on¹⁴C-labeled production of nornicotine to calculate nicotine demethylase activity.

Microsomal preparations from control yeast cells expressing CYP71D20 did not have any detectable microsomal nicotine demethylase activity. In contrast, microsomal samples obtained from yeast cells expressing the tobacco nicotine demethylase gene show significant levels of nicotine demethylase activity. NADPH is required for nicotine demethylase activity and was shown to be inhibited by p450 specific inhibitors, consistent with tobacco nicotine demethylase as p 450. The enzymatic activity of tobacco nicotine demethylase (D121-AA8) was approximately 10.8pkat/mg protein as calculated from the radioactive intensity and protein concentration. A typical set of enzyme assay results obtained for yeast cells is shown in Table 1.

Table 1: demethylase Activity in microsomes of D121-AA8 and control P450 expressing Yeast cells

^*Average results of 3 replicates

Together, these experiments demonstrate that cloned full-length tobacco nicotine demethylase (SEQ ID NO: 3; D121-AA8) encodes a cytochrome p450 protein that, when expressed in yeast, catalyzes the conversion of nicotine to nornicotine.

Example 11

Related amino acid sequence identity of isolated nucleic acid fragments

The amino acid sequence of the nucleic acid sequence of the cytochrome p450 fragment obtained in example 8 was deduced. The putative region corresponds to the amino acid to carboxy-terminal end, or stop codon, immediately following the GXRXCP (A/G) (SEQ ID NO: 13) sequence motif. After comparing the sequence identity of the fragments, a unique set of those sequences with 70% or greater amino acid identity was observed. It was observed that the preferred group was those sequences having 80% or more amino acid identity, the more preferred group was those having 90% or more amino acid identity, and the most preferred group was those having 99% or more amino acid identity. Several unique nucleic acid sequences were found to have complete amino acid identity with other fragments, so only one member with the same amino acid was reported.

At least one member of each amino acid identity group was selected for gene cloning and functional studies using plants. In addition, panel members were selected for gene cloning and functional studies, said members being assessed by, e.g., RNA and DNA analysis, and being differentially affected by ethylene treatment or other biological differences. To facilitate gene cloning, expression studies, and overall plant evaluation, peptide-specific antibodies can be prepared based on sequence identity and different sequences.

Example 12

Related amino acid sequence identity for full-length clones

The nucleic acid sequences of the full-length nicotiana genes cloned in example 5 were deduced for their complete amino acid sequences. Cytochrome P450 genes were identified by the presence of three conserved P450 domain motifs corresponding to UXXRXXXZ (SEQ ID NO: 14), PXRFXF (SEQ ID NO: 15) or GXRXC (SEQ ID NO: 16) on the carboxy terminus, where U is E or K, X is any amino acid, and Z is P, T, S or M. All p450 genes were characterized for amino acid identity by comparing their full-length sequences to each other and to known tobacco genes using the BLAST program. The program uses the NCBI special BLAST tool (align two sequences (bl2seq),http://www.ncbi.nlm.nih.gov/blast/ bl2seq/bl2.html). The two sequences were aligned under BLASTN without filters for nucleic acid sequences and BLASTP for amino acid sequences. Based on their percent amino acid identity, each sequence is classified into an identity group, wherein the group comprises members that share at least 85% identity with another member. It is observed that the preferred group is those sequences having 90% or more amino acid identity, the more preferred group is those sequences having 95% or more amino acid identity, and the most preferred group is those sequences having 99% or more amino acid identity. Inferring the amino acid sequence of the full-length nicotine demethylase gene to have seq id NO: 4 (fig. 3).

Example 13

Nicotiana cytochrome P450 clones lacking one or more tobacco P450-specific domains

Four clones had high nucleic acid homology, ranging from 90% to 99% nucleic acid homology. However, due to nucleotide frameshifting, these genes do not contain one or more of the three C-terminal cytochrome p450 domains and are excluded from the identity group.

Example 14

Application of nicotiana cytochrome P450 segment and clone in tobacco quality change regulation

The use of tobacco p450 nucleic acid fragments or intact genes is useful in identifying and selecting those plants that have altered tobacco phenotypes or tobacco components, and more importantly, altered metabolites. Transgenic tobacco plants are produced by various transformation systems that incorporate a nucleic acid fragment or full-length gene selected from those reported herein in a down-regulated orientation, e.g., antisense orientation, or over-expression, e.g., sense orientation, etc. For overexpression of the full-length gene, any nucleic acid sequence encoding the complete or functional part or amino acid sequence of the full-length gene according to the invention is advantageous. These nucleic acid sequences are ideally effective in increasing the expression of an enzyme and thus result in phenotypic effects within nicotiana. Homozygous nicotiana lines are obtained by a series of backcrosses and evaluated for phenotypic changes, including, but not limited to, analysis of endogenous p450 RNA, transcripts, p450 expression peptides, and plant metabolite concentrations using techniques routinely available to those of ordinary skill in the art. The changes exhibited in the tobacco plant provide information on the functional effect of the selected gene of interest or for use as information on the preferred nicotiana species.

Example 15

Cloning of genomic tobacco nicotine demethylase from transformant Burley tobacco

Genomic DNA was extracted from the transformant Burley tobacco Plant line 4407-33 (tobacco variety 4407 line) using the Qiagen Plant Easy kit as described in the above examples (see also manufacturer's methods).

Primers were designed based on the 5 'promoter and 3' UTR regions cloned in the previous examples. The forward primers were 5'-GGC TCTAGA TAA ATC TCT TAA GTT ACT AGG TTC TAA-3' (SEQ ID NO: 17) and 5'-TCT CTA AAG TCCCCT TCC-3' (SEQ ID NO: 25) and the reverse primers were 5'-GGC TCT AGA AGT CAA TTA TCT TCT ACAAAC CTT TATATA TTA GC-3' (SEQ ID NO: 18), and 5'-CCA GCA TTC CTC AAT TTC-3' (SEQ ID NO: 26). PCR was applied to 4407-33 genomic DNA using 100. mu.l of the reaction mixture. PCR amplification was performed using Pfx high fidelity enzyme. After electrophoresis, the PCR product was observed on a 1% agarose gel. A single band with a molecular weight of approximately 3.5kb was observed and excised from the gel. The resulting band was purified using a gel purification kit (Qiagen; based on the manufacturer's protocol). The purified DNA was digested by the enzyme Xba I (NEB; used according to the manufacturer's instructions). The pBluescript plasmid was digested by Xba I using the same method. The fragments were gel purified and ligated into pBluescript plasmid. The ligation mixture was transformed into competent cells GM1O9 and plated onto LB plates containing 100mg/l ampicillin, with a blue/white screen. White colonies were picked and cultured in 10ml of LB liquid medium containing ampicillin. DNA was extracted by miniprep. Plasmid DNA containing the insert was sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, California) based on the manufacturer's protocol. Sequencing was performed using the T3 and T7 primers, as well as 8 other inner primers. The sequences were combined and analyzed, thereby providing a genomic sequence (SEQ ID NO: 1; FIGS. 2-1 to 2-3).

Converting SEQ ID NO:1 and SEQ ID NO: 3 (identified as the sequence of SEQ ID NO: 5; FIG. 4) enables the determination of a single intron within the coding portion of the gene. As shown in figure 1, the genomic structure of tobacco nicotine demethylase comprises two exons flanking a single intron. The first exon spans SEQ ID NO:1, which encodes the amino acid sequence of SEQ id no: 2, and a second exon spanning amino acids 1-313 of SEQ ID NO:1, which encodes the nucleotide sequence of SEQ ID NO: 2, amino acid 314-. Thus, the intron spans SEQ ID NO: nucleotide 2950 of 1 and 3946. The intron sequence is provided in fig. 4 and is SEQ ID NO: 5. The translation product of the genomic DNA sequence is represented as SEQ ID NO: 2 is provided in fig. 2-1. The tobacco nicotine demethylase amino acid sequence comprises an endoplasmic reticulum membrane anchoring motif.

Example 16

Cloning of the 5 'flanking sequence (SEO ID NO: 6) and the 3' UTR (SEQ ID NO: 7) from the transformant tobacco

A.Isolation of Total DNA from transformant tobacco leaf tissue

Genomic DNA was isolated from leaves of the transformant tobacco 4407-33. Isolation of DNA was performed according to the manufacturer's protocol using the DNeasy Plant Mini kit from Qiagen, Inc (Valencia, Ca). The manufacturer's manuals Dneasy' Plant Mini and DNeasy Plant Maxi Handbook, Qiagen January 2004 are incorporated herein by reference. The DNA preparation method comprises the following steps: tobacco leaf tissue (approximately 20mg dry weight) was ground to a fine powder under liquid nitrogen for 1 minute. Tissue powder was transferred into 1.5ml tubes. Buffer AP1 (400. mu.l) and 4. mu.l of RNase stock solution (100mg/ml) were added to a maximum of 100mg of ground leaf tissue and vortexed vigorously. The mixture was incubated at 65 ℃ for 10min and mixed 2-3 times during the incubation by inverting the tube. Buffer AP2 (130. mu.l) was then added to the lysate. The mixture was mixed and incubated on ice for 5 min. Lysates were applied to a QIAshreder Mini spin column and centrifuged for 2min (14,000 rpm). The flow-through fraction was transferred to a new tube without disturbing the cell debris pellet. Buffer AP3/E (1.5 volumes) was then added to the clarified lysate and mixed by aspiration. The mixture (650 μ l) from the previous step including any precipitate was applied to a DNeasy Mini spin column. The mixture was centrifuged at > 6000Xg (> 8000rpm) for 1min and the flow through was discarded. This was repeated with the remaining sample and the flow through and collection tubes discarded. The DNeasy Mini spin column was placed in a new 2ml collection tube. Buffer AW (500. mu.l) was then added to the DNeasy column and centrifuged for 1min (> 8000 rpm). The flow through was discarded. The collection tube is reused in the next step. Buffer AW (500. mu.l) was then added to the DNeasy column and centrifuged for 2min (> 14,000rpm) to dry the membrane. The DNeasy column was transferred to a 1.5ml tube. Buffer AE (100 μ l) was then pipetted onto the DNeasy membrane. The mixture was incubated at room temperature (15-25 ℃) for 5min and then centrifuged for 1min (> 8000rpm) to elute.

The quality and quantity of DNA was assessed by running the samples on agarose gels.

B.Cloning of the 5' flanking sequence of the structural Gene

Cloning of a nucleic acid sequence from SEQ ID NO:1, or a structural gene of 1. First, an appropriate restriction enzyme is selected based on the restriction site in the fragment of known sequence and the spacing of the restriction sites downstream of the 5' flanking sequence. Two primers were designed based on this known fragment. The forward primer is located downstream of the reverse primer. The reverse primer is located in the 3' portion of the known fragment.

The cloning method comprises the following steps:

purified genomic DNA (5. mu.g) was digested with 20-40 units of the appropriate restriction enzymes (EcoRI and SpeI) in a 50. mu.l reaction mixture. Agarose gel electrophoresis was performed with 1/10 volumes of the reaction mixture to determine if the DNA was completely digested. Direct ligation was performed by overnight ligation at 4 ℃ after complete digestion. 200 μ l of a reaction mixture containing 10 μ l of digested DNA and 0.2 μ l of T4DNA ligase (NEB) was ligated overnight at 4 ℃. PCR of the ligation reaction was performed after obtaining the artificial small circular genome. PCR was performed in 50. mu.l reaction mixture in two different orientations using 10. mu.l ligation reaction and 2 primers from known fragments. A gradient PCR procedure was applied with an annealing temperature of 45-56 ℃.

Agarose gel electrophoresis was performed to check the PCR reaction. The desired band was cut from the gel and purified using the QIAquick gel purification kit from QIAGEN. The purified PCR fragment was ligated into pGEM-T Easy vector (Promega, Madison, Wis.) according to the manufacturer's instructions. The transformed DNA plasmid was extracted by miniprep using the SV miniprep kit (Promega, Madison, Wis.) according to the manufacturer's instructions. Plasmid DNA containing the insert was sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, Calif.). The 5' flanking sequence of approximately 758nt (nucleotide 1241-2009 of SEQ ID NO: 1) was cloned by the method described above.

C.Cloning of the longer 5' flanking sequence of the structural Gene (SEQ ID NO: 6; FIG. 5)

The BD genome Walker general kit (Clontech laboratories, Inc., Palo alto, Calif.) was used to clone the structural gene, the additional 5 'flanking sequence of D121-AA8, according to the manufacturer's user manual. The manufacturer's manual BD GenomeWalker August, 2004 is incorporated herein by reference. The size and purity of tobacco genomic DNA was tested by subjecting the samples to electrophoresis on 0.5% agarose gel. A total of 4 blunt-end reactions (DRA I, STU I, ECOR V, PVU II) were set up for the tobacco 33 library genome walking construction. After purification of the digested DNAs, the digested genomic DNAs are ligated to a genomic walking linker. The four digested DNAs were subjected to preliminary PCR reaction by using adaptor primer AP1 and gene specific primer (CTCTATTGATACTAGCTGGTTTTGGAC; SEQ ID NO: 19) from D121-AA 8. Nested PCR was performed using the preliminary PCR product directly as a template. The adaptor nested primers provided by the kit and nested primers (GGAGGGAGAGTATAACTTACGGATTC; SEQ ID NO: 20) from the known clone D121-AA8(SEQ ID NO: 3) were used in the PCR reaction. The PCR product was detected by performing gel electrophoresis. The desired band was cut from the gel and the PCR fragment was purified using the QIAquick gel purification kit from QIAGEN. The purified PCR fragment was ligated into pGEM-T Easy vector (Promega, Madison, Wis.) according to the manufacturer's instructions. The transformed DNA plasmid was extracted by miniprep using the SV miniprep kit (Promega, Madison, Wis.) and following the manufacturer's instructions. Plasmid DNA containing the insert was sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, Calif.). Another 5' flanking sequence of approximately 853nt was cloned by the method described above, including SEQ ID NO: nucleotide 399 of 1 and 1240.

A second round of genome walking was performed according to the same method except that the following primers GWR1A (5'-AGTAACCGATTGCTCACGTTATCCTC-3') (SEQ ID NO: 21) and GWR2A (5'-CTCTATTCAACCCCACACGTAACTG-3') (SEQ ID NO: 22) were used. An additional about 398nt flanking sequence was cloned by this method, including SEQ ID NO:1 from nucleotide 1 to 398.

A search for regulatory elements revealed that, in addition to the "TATA" box, the "CAAT" box, and the "GAGA" box, several MYB-like recognition sites and organ-specific elements are present in the tobacco nicotine demethylase promoter region. Putative derivative (elicitritor) reactive and nitrogen-regulated elements identified using the same method also appear in the promoter region.

D.Cloning of the 3' flanking sequence of the structural Gene

The BD genome walking universal kit (Clontech laboratories, inc., PaloAlto, CA) was used to clone the 3 'flanking sequence of the structural gene D121-AA8 according to the manufacturer's instruction manual. The cloning procedure was the same as described in section C above of this example, except for the gene-specific primers used. The first primer was designed near the end of the D121-AA8 structural gene (5'-CTA AAC TCT GGT CTG ATC CTG ATA CTT-3') (SEQ ID NO: 23). A nested primer was further designed downstream (CTA TAC GTA AGG TAA ATC CTG TGGAAC) of primer 1 of the D121-AA8 structural gene (SEQ ID NO: 24). The final PCR product was checked by gel electrophoresis. The desired tape was cut from the gel. The PCR fragment was purified using QIAquick gel purification kit from QIAGEN. The purified PCR fragment was ligated into pGEM-T Easy vector (Promega, Madison, Wis.) according to the manufacturer's instructions. The transformed DNA plasmid was extracted by miniprep using the SV miniprep kit (Promega, Madison, Wis.) according to the manufacturer's instructions. Plasmid DNA containing the insert was sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, Calif.). An additional 3' flanking sequence of about 1617 nucleotides (nucleotides 4731-6347 of SEQ ID NO: 1) was cloned by the method described above. The nucleic acid sequence of the 3' UTR region is shown in figure 6.

WO 03/078577, WO 2004/035745, PCT/US/2004/034218, and PCT/US/2004/034065 and all other references, patents, patent application publications, and patent applications referenced herein are hereby incorporated by reference to the same extent as if each such reference, patent application publication, and patent application was individually incorporated by reference.

It is contemplated that modifications and improvements will occur to those skilled in the art in the practice of the invention, upon consideration of the foregoing detailed description of the invention. Accordingly, such modifications and improvements are intended to be included within the scope of the following claims.

Claims (12)

1. An expression vector comprising a nucleic acid consisting of SEQ ID NO. 1, said vector being capable of directing the expression of said nucleic acid.

2. A processed tobacco plant component from a transgenic tobacco plant comprising a vector comprising the nucleotide sequence of SEQ ID No. 1, said processed tobacco plant component having a reduced level of nornicotine or N' -nitrosonornicotine relative to the level in a corresponding processed tobacco plant component obtained from a wild-type non-transformed tobacco plant.

3. The processed tobacco plant component of claim 2, wherein said processed tobacco plant component is tobacco lamina.

4. The processed tobacco plant component of claim 3, wherein said processed tobacco plant component has a reduced level of nornicotine relative to the level in a processed tobacco plant component obtained from a wild-type non-transformed tobacco plant.

5. The processed tobacco plant component of claim 3, wherein the processed tobacco plant component is from dark tobacco, burley tobacco, flue-cured tobacco, dark air-cured tobacco, or oriental tobacco.

6. A tobacco product comprising the processed tobacco plant component of claim 3.

7. The tobacco product of claim 6, wherein said tobacco product is smokeless tobacco.

8. The tobacco product of claim 7, wherein said tobacco product is wet or dry snuff.

9. The tobacco product of claim 7, wherein said tobacco product is chewing tobacco.

10. The tobacco product of claim 6, wherein said tobacco product is a cigarette.

11. The tobacco product of claim 6, wherein said tobacco product is a cigar.

12. The tobacco product of claim 6, wherein said tobacco product is a cigarillo.