ES2373881T3 - GENOMIC CLONY OF TOBACCO NICOTINE DESMETILASE AND USES OF THE SAME. - Google Patents

Abstract

Isolated nucleic acid molecule encoding a nicotine demethylase, said nucleic acid molecule (a) comprising the sequence of SEQ ID NO: 1, or (b) 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.

Description

Genomic clone of tobacco nicotine demethylase and uses thereof

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

Background

Cytochrome p450 catalyzes enzymatic reactions for a diverse range of chemically distinct substrates that include oxidative, peroxidative and reductive metabolism of xenobiotic and endogenous substrates. In plants, p450 participates in biochemical pathways that include the synthesis of plant products such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides and glucosylonates (Chappell, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 521-547, nineteen ninety five). Cytochrome p450s, also known as hemo-thiolate p450 proteins, usually acts as terminal oxidases in multi-component electron transfer chains, called p450-containing monooxygenase systems. Specific reactions catalyzed by these enzyme systems include demethylation, hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S- and Odesalkylations, desulphation, deamination and reduction of N-oxide, nitro and azo groups.

The diverse role of the p450 enzymes of the Nicotiana plant has been implicated in the generation of a variety of plant metabolites such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, glucosylonates and a large number of other chemical entities. Some p450 enzymes may have an impact on the composition of plant metabolites. For example, it has long been desired to improve the taste and aroma of certain plants by altering the profile of a plant of selected fatty acids through cultivation; however, very little is known about the mechanisms involved in controlling the levels of these leaf constituents. Regulation by decrease or regulation by increase of p450 enzymes associated with the modification of fatty acids can facilitate the accumulation of desired fatty acids that provide more preferred phenotypic qualities of the leaf.

The function of p450 enzymes and their increasing roles in plant constituents are still being discovered. For example, it was found that a special class of p450 enzymes catalyzed the breakdown of fatty acid into volatile C6 and C9 aldehydes and aldehydes that are major contributors to the "fresh green" smell of fruits and vegetables. The level of other p450 selected as novel target can be altered to enhance the qualities of the leaf constituents by modifying the lipid composition and related decomposition metabolites in the Nicotiana leaf. Several of these constituents in the leaf are affected by the senescence that stimulates the maturation of quality properties of the leaf. Still other reports have shown that p450 enzymes play a functional role in the alteration of fatty acids that are involved in disease resistance and plant-pathogen interactions.

In other examples, it has been suggested that p450 enzymes are involved in alkaloid biosynthesis. As provided in the patent applications by the applicant, of which the present application claims priority, nornicotine, a minor alkaloid found in Nicotiana tabacum, is produced by p450-mediated demethylation of nicotine followed by acylation and nitrosation in the N position thereby producing a series of N-acylnonicotines and N-nitrosonomicotines. It is believed that N-demethylation, catalyzed by a p450 demethylase, is a primary source of nornicotine biosynthesis in Nicotiana.

There is a need in the art of reagents and methods to modify plant phenotypes. In particular, there is a need for reagents and methods to modify nicotine demethylase. The present invention provides several strategies for modifying the expression of a nicotine demethylase.

Summary of the invention

The present inventors have identified and characterized a genomic clone of nicotine demethylase from tobacco. Sequences are included herein for the coding region of the nicotine demethylase protein, the 3 'untranslated region ("UTR in 3'"), an individual intron and the promoter of the nicotine demethylase gene together with their regulatory sequences of the transcript (figure 1). The use of these sequences to create transgenic plants that have altered levels of nornicotine or N’-nitrosonomicotine (“NNN”) or both with respect to a control plant is also described.

Accordingly, the invention describes an isolated nucleic acid molecule, a transgenic tobacco plant, a plant component of the tobacco plant, a tobacco product, a seed of the tobacco plant, a recombinant tobacco nicotine demethylase and various methods. which imply the same, as defined in the claims.

Accordingly, in the first aspect, the description describes an isolated nucleic acid molecule, for example, a DNA sequence, which contains 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 containing a nucleotide sequence that is at least 70% identical to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or that contains nucleotides 2010-2949 and / or 3947-4562 of SEQ ID NO: 1, or that contains the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. The isolated nucleic acid molecule of the first aspect of the description, for example, is operably linked to a functional promoter in a plant cell and desirably is contained in an expression vector. In other desirable embodiments, the expression vector is contained in a cell, for example, a plant cell. Desirably, the plant cell, such as a plant cell of tobacco, is included in a plant. In another desirable embodiment, the description describes a seed, for example, a tobacco seed, from a plant containing the expression vector, in which the seed includes an isolated nucleic acid molecule that hybridizes under stringent conditions with the sequence of SEQ ID NO: 3 operatively linked to a heterologous promoter sequence. In addition, the description describes a plant derived from a germinated seed containing the expression vector, a leaf, either green or cured, of the plant, and a manufacturing article prepared from the leaf.

In another desirable embodiment, the nucleotide sequence contains a sequence that hybridizes under stringent conditions with the complement of the nucleotide sequence of SEQ ID NO: 1 and / or SEQ ID NO: 3, or to a fragment of SEQ ID NO: 1 I o SEQ ID NO: 3. Desirably, the nucleotide sequence encodes a nicotine demethylase that is substantially identical to the amino acid sequence of SEQ ID NO: 2. In a further desirable embodiment of the first aspect of the invention, nicotine demethylase has an amino acid sequence identity of at least 70% with the amino acid sequence of nicotine demethylase of SEQ ID NO: 2 or with a fragment of a nicotine demethylase having altered enzymatic activity (for example, reduced) compared to the full length polypeptide. Desirably, nicotine demethylase includes the amino acid sequence of SEQ ID NO: 2.

In another aspect, the description describes an isolated nucleic acid molecule that contains a promoter that hybridizes under stringent conditions with the sequence of SEQ ID NO: 6, or a fragment thereof that directs transcription. Desirably, the promoter (i) is induced after treatment with ethylene or during senescence; and (ii) includes (a) base pairs 1-2009 of SEQ ID NO: 1, or (b) at least 200 consecutive base pairs identical to 200 consecutive base pairs of the sequence defined by base pairs 1 -2009 of SEQ ID NO: 1, or (c) a nucleotide part of 20 base pairs identical in sequence to a part of 20 consecutive base pairs of the sequence set forth in base pairs 1-2009 of SEQ ID NO :one.

A further aspect of the description describes an isolated nucleic acid promoter containing a nucleotide sequence having a sequence identity of 50% or more with the sequence of SEQ ID NO: 6. Desirably, this isolated nucleic acid promoter is induced after treatment with ethylene or during senescence and, for example, includes the sequence of SEQ ID NO: 6. Alternatively, the promoter may include a fragment that can be obtained from SEQ ID NO: 6, in which the fragment directs the transcription of a heterologous gene or reduces or alters the enzymatic activity of nicotine demethylase (eg, silences expression gene) In a desirable embodiment, the promoter sequence is operably linked to a heterologous nucleic acid sequence, and may, for example, be contained in an expression vector. In other desirable embodiments, the expression vector is contained in a cell, for example, a plant cell. Desirably, the plant cell, such as a plant cell of tobacco, is included in a plant. In another desirable embodiment, the description describes a seed, for example, a tobacco seed, of a plant containing the expression vector, in which the seed includes an isolated nucleic acid molecule that hybridizes under stringent conditions with the sequence of SEQ ID NO: 6 operably linked to a heterologous nucleic acid sequence. In addition, the description describes a plant derived from a germinated seed containing the promoter of this aspect of the description, a leaf, either green or cured, of the plant, and a manufacturing article prepared from the leaf.

Another aspect of the description describes a method of expressing a heterologous gene in a plant. This method involves (i) introducing into a plant cell a vector containing a promoter sequence having a sequence identity of 50% or more with the sequence of SEQ ID NO: 6 operably linked to a heterologous nucleic acid sequence; and (ii) regenerate a plant from the cell. In addition, this method may involve the sexual transmission of the vector to the progeny and, in addition, may include the stage of collecting the seed produced by the progeny.

In yet another aspect, the description describes a method of reducing the expression of nicotine demethylase in a tobacco plant. This method includes the steps of (i) introducing into the tobacco plant a vector containing the sequence of SEQ ID NO: 6 or a fragment that can be obtained from SEQ ID NO: 6 operably linked to a heterologous nucleic acid sequence and (ii) express the vector in the tobacco plant. In a desirable embodiment of this method, the expression of nicotine demethylase is silenced. In another desirable embodiment, the vector expresses RNA, such as antisense RNA or an RNA molecule that can induce RNA interference (RNAi).

In a further desirable aspect, the description describes an isolated nucleic acid molecule containing an intron that hybridizes under stringent conditions with the sequence of SEQ ID NO: 5, or a fragment thereof that reduces or alters the enzymatic activity of nicotine demethylase (for example, silences gene expression) or can serve as a molecular marker to identify nucleic acid sequences of nicotine demethylase. In a desirable embodiment, the intron includes (a) base pairs 2950-3946 of SEQ ID NO: 1, or (b) at least 200 consecutive base pairs identical to the 200 consecutive base pairs of the sequence defined by the base pairs 2950-3946 of SEQ ID NO: 1, or (c) a nucleotide part of 20 base pairs identical in sequence to a part of 20 consecutive base pairs of the sequence set forth in base pairs 2950-3946 of SEQ ID NO: 1.

Another desirable aspect of the description describes an isolated nucleic acid intron that includes a nucleotide sequence having a sequence identity of 50% or more with the sequence of SEQ ID NO: 5, or a fragment thereof that reduces or alters The enzymatic activity of nicotine demethylase (for example, silences gene expression) or can serve as a molecular marker to identify nucleic acid sequences of nicotine demethylase. Silencing gene expression may involve, for example, homologous recombination or a mutation that results in a gene product that has no nicotine demethylase activity. In particular, the intron can include the sequence of SEQ ID NO: 5 or a fragment that can be obtained from SEQ ID NO: 5. Desirably, an isolated nucleic acid molecule that includes an intron is operably linked to a heterologous nucleic acid sequence and this sequence is desirably included in an expression vector. In another embodiment, the expression vector is contained in a cell, such as a plant cell. In particular, the cell can be a tobacco cell. A plant, for example, a tobacco plant, which includes a plant cell that contains the sequence of SEQ ID NO: 5 or a fragment that can be obtained from SEQ ID NO: 5 operably linked to a heterologous nucleic acid sequence in An expression vector is another desirable embodiment of the present invention. In addition, a seed, for example, a tobacco seed, of a plant, in which the seed contains an intron that hybridizes under stringent conditions with SEQ ID NO: 5 operatively linked to a heterologous nucleic acid sequence is also desirable. In addition, the description describes a plant derived from the germinated seed that contains the intron of this aspect of the invention, a leaf, either green or cured, of the plant, and a manufacturing article prepared from the green leaf or cured

An additional aspect of the description describes a method of expressing an intron in a plant. This method involves (i) introducing into an plant cell an expression vector containing the sequence of SEQ ID NO: 5 or a fragment that can be obtained from SEQ ID NO: 5 operably linked to a heterologous nucleic acid sequence; and (ii) regenerate a plant from the cell. In a desirable embodiment, this method also involves

(iii) sexually transmit the vector to the progeny, and may include the additional stage of collecting the seed produced by the progeny. The method desirably includes, for example, regenerating a plant from the germinated seed, a leaf, either green or cured, from the plant, and a method of preparing a manufacturing article from the leaf.

In yet another aspect, the description describes a method of reducing the expression of nicotine demethylase in a tobacco plant. This method includes the steps of (i) introducing into the tobacco plant a vector containing the sequence of SEQ ID NO: 5 or a fragment that can be obtained from SEQ ID NO: 5 operably linked to a heterologous nucleic acid sequence and (ii) express the vector in the tobacco plant. In a desirable embodiment of this method, the expression of nicotine demethylase is silenced. In another desirable embodiment, the vector expresses RNA, such as antisense RNA or an RNA molecule that can induce RNA interference (RNAi).

In a further aspect, the description describes an isolated nucleic acid molecule that contains a non-translated region that hybridizes under stringent conditions with the sequence of SEQ ID NO: 7 or a fragment thereof that can alter the expression pattern of a gene , reduces or alters the enzymatic activity of nicotine demethylase (for example, silences gene expression), or can be used as a marker to identify nucleic acid sequences of nicotine demethylase. In a desirable embodiment of this aspect of the description, the untranslated region includes (a) base pairs 4563-6347 of SEQ ID NO: 1, or (b) at least 200 consecutive base pairs identical to the 200 pairs of consecutive bases of the sequence defined by base pairs 45636347 of SEQ ID NO: 1, or (c) a nucleotide part of 20 base pairs identical in sequence to a part of 20 consecutive base pairs of the sequence set forth in the base pairs 4563-6347 of SEQ ID NO: 1.

A further desirable aspect of the description describes an untranslated region of isolated nucleic acid containing a nucleotide sequence having a sequence identity of 50% or more with the sequence of SEQ ID NO: 7. Desirably, the untranslated region includes the sequence of SEQ ID NO: 7 or the untranslated region includes a fragment that can be obtained from SEQ ID NO: 7 that can alter the expression pattern of a gene, reduce or alter activity Nicotine demethylase enzyme (for example, silences gene expression), or can be used as a marker to identify nicotine demethylase nucleic acid sequences. The untranslated region is desirably operably linked to a heterologous nucleic acid sequence and may be contained in an expression vector. In addition, this expression vector is desirably contained in a cell, such as a plant cell, for example, a tobacco cell. Another desirable embodiment of the description describes a plant, such as a tobacco plant, that includes a plant cell that contains a vector that includes an isolated nucleic acid sequence that has a sequence identity of 50% or more with the SEQ sequence. ID NO: 7 and is operably linked to a heterologous nucleic acid sequence.

The description also describes a seed, for example, a tobacco seed, of a plant, in which the seed includes a non-translated region that hybridizes under stringent conditions with SEQ ID NO: 7 operatively linked to a heterologous nucleic acid sequence. In addition, the description describes a plant derived from a germinated seed containing the untranslated region of this aspect of the invention, a leaf, either green or cured, of the plant, and a manufacturing article prepared from the leaf. green or cured

In a further aspect, the description describes a method of expressing a region not translated in a plant. This method involves (i) introducing into a plant cell a vector containing an isolated nucleic acid sequence that has a sequence identity of 50% or more with the sequence of SEQ ID NO: 7 and is operably linked to an acid sequence heterologous nucleic; and (ii) regenerate a plant from the cell. In addition, this method may also involve (iii) sexually transmitting the vector to the progeny, and desirably, includes the additional step of collecting the seed produced by the progeny. The method desirably includes regenerating a plant from the germinated seed, a leaf, either green or cured, from the plant, and a method of preparing a manufacturing article prepared from the green or cured leaf.

In addition, the description describes a method of reducing the expression or alteration of the enzymatic activity of nicotine demethylase in a tobacco plant. This method includes the steps of (i) introducing into the tobacco plant a vector containing an isolated nucleic acid sequence that has a sequence identity of 50% or more with the sequence of SEQ ID NO: 7 and is operably linked to a heterologous nucleic acid sequence and

(ii) express the vector in the tobacco plant. Desirably, the expression of nicotine demethylase is silenced. In other desirable embodiments, the vector expresses RNA, for example, antisense RNA or an RNA molecule that can induce RNA interference (RNAi).

Another aspect of the description describes an expression vector that includes a nucleic acid molecule that contains a nucleotide sequence encoding a nicotine demethylase, in which the vector can direct the expression of nicotine demethylase encoded by the nucleic acid molecule. isolated. Desirably, the vector includes the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In other desirable embodiments, the description describes a plant or plant component, for example, a tobacco plant or plant component (for example, a tobacco stem or leaf), which includes a nucleic acid molecule that contains a nucleotide sequence that encodes a polypeptide that demethylates nicotine.

A further aspect of the description describes a cell that contains an isolated nucleic acid molecule that includes a nucleotide sequence that encodes a nicotine demethylase. Desirably this cell is a plant cell or a bacterial cell, such as an Agrobacterium.

Another aspect of the description describes a plant or plant component (for example, a tobacco stem or leaf) that contains an isolated nucleic acid molecule encoding a nicotine demethylase, in which the nucleic acid molecule is expressed in the plant. or the plant component. Desirably, the plant or plant component is an angioesperm, a dicot, a solanum plant or a species of Nicotiana. Other desirable embodiments of this aspect are a seed or a plant cell or plant component, as well as a leaf, either green or cured, derived from the plant and a manufacturing article prepared therefrom.

In a further aspect, the description describes a tobacco plant that has reduced expression of a nucleic acid sequence encoding a polypeptide, for example, one that includes the sequence of SEQ ID NO: 2, and that demethylates nicotine, in which reduced expression (or a reduction in enzymatic activity) reduces the level of nornicotine in the plant. In a desirable embodiment, the tobacco plant is a transgenic plant, such as one that includes a transgene that, when expressed in the transgenic plant, silences the gene expression of an endogenous tobacco nicotine demethylase.

In particular, the transgenic plant desirably includes one or more of the following: a transgene that expresses an antisense molecule of a tobacco nicotine demethylase or an RNA molecule that can induce RNA interference (RNAi); a transgene that, when expressed in the transgenic plant, jointly suppresses the expression of a tobacco nicotine demethylase; a transgene encoding a negative dominant gene product, for example, a mutated form of the amino acid sequence of SEQ ID NO: 2; a point mutation in a gene that codes for the amino acid sequence of SEQ ID NO: 2; a deletion in a gene that codes for a tobacco nicotine demethylase; and an insertion in a gene that codes for a nicotine demethylase tobacco.

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

Another aspect of the description describes a tobacco plant containing a recombinant expression cassette stably integrated into the genome thereof, in which the cassette can effect a reduction in nicotine demethylase activity. The seeds of this tobacco plant are described in a desirable embodiment. Other desirable embodiments include a leaf, either green or cured, derived from this plant and a manufacturing article prepared therefrom.

A further aspect of the description describes a method of expression of a tobacco nicotine demethylase in a plant. This method involves (i) introducing into an plant cell an expression vector that includes a nucleic acid molecule that contains a nucleotide sequence encoding a nicotine demethylase; and (ii) regenerate a plant from the cell. In a desirable embodiment, this method describes the sexual transmission of the vector to the progeny, and desirably also includes the additional step of collecting the seed produced by the progeny. Additional desirable embodiments include a plant derived from the germinated seed, a leaf, either green or cured, from the plant, and a manufacturing article prepared from the green or cured leaf.

A further aspect of the description describes a substantially pure tobacco nicotine demethylase. Desirably, this tobacco nicotine demethylase includes an amino acid sequence that has an identity of at least 70% with the amino acid sequence of SEQ ID NO: 2 or includes the amino acid sequence of SEQ ID NO: 2. In a desirable embodiment, tobacco nicotine demethylase, after expression in a plant cell, converts nicotine to nornicotine. In other desirable embodiments, tobacco nicotine demethylase, after expression in a plant cell, is predominantly located in the leaves, or tobacco nicotine demethylase is induced by ethylene or expressed during senescence of the plant.

In a further aspect, the description describes a substantially pure antibody that specifically recognizes and binds to a tobacco nicotine demethylase. Desirably, the antibody recognizes and binds to a recombinant tobacco nicotine demethylase, for example, one containing the sequence of SEQ ID NO: 2 or a fragment thereof.

Another aspect of the description describes a method of producing a tobacco nicotine demethylase. This method involves the steps of: (a) providing a cell transformed with an isolated nucleic acid molecule that contains a nucleotide sequence encoding a polypeptide that demethylates nicotine; (b) culturing the transformed cell under conditions to express the isolated nucleic acid molecule; and (c) recover tobacco nicotine demethylase. The description also describes a recombinant tobacco nicotine demethylase produced according to this method.

In a further aspect, the description describes a method of isolating a tobacco nicotine demethylase or fragment thereof. This method involves the steps of: (a) contacting the nucleic acid molecule of SEQ ID NOS: 1, 3, 5, 6 or 7 or a part thereof with a nucleic acid preparation from a plant cell under hybridization conditions that provide for the detection of nucleic acid sequences that have a sequence identity of at least 70% or greater with the nucleic acid sequence of SEQ ID NOS: 1, 3, 5, 6 or 7; and (b) isolate hybridizing nucleic acid sequences.

In a further aspect, the description describes another method of isolating a tobacco nicotine demethylase or fragment thereof. This method includes the steps of: (a) providing a sample of plant cell DNA;

(b) providing a pair of oligonucleotides that have sequence identity with a region of a nucleic acid molecule that has the sequence of SEQ ID NOS: 1, 3, 5, 6 or 7; (c) contacting the oligonucleotide pair with the plant cell DNA under conditions suitable for DNA amplification mediated by the polymerase chain reaction; and (d) isolate the amplified tobacco nicotine demethylase or fragment thereof. In a desirable embodiment of this aspect, the amplification step is carried out using a cDNA sample prepared from a plant cell. In another desirable embodiment, tobacco nicotine demethylase encodes a polypeptide that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2.

A further aspect of the description describes a method to reduce the expression of tobacco nicotine demethylase in a plant or plant component. This method involves the steps of: (a) introducing into plant cells a transgene encoding a tobacco nicotine demethylase operably linked to a functional promoter in plant cells to provide transformed plant cells; and (b) regenerating a plant or plant component from the transformed plant cells, in which tobacco nicotine demethylase is expressed in the cells of the plant or plant component, thereby reducing the expression of tobacco nicotine demethylase in a plant or plant component. In particular embodiments of this aspect of the description, the transgene encoding tobacco nicotine demethylase is constitutively expressed or induciblely expressed, for example, in a tissue-specific, cell-specific or organ-specific manner. In another embodiment of this aspect of the description, the expression of the transgene together suppresses the expression of an endogenous tobacco nicotine demethylase.

A further aspect of the description describes another method to reduce the expression of tobacco nicotine demethylase in a plant or plant component. This method includes the steps of: (a) introducing into plant cells a transgene that encodes an antisense coding sequence of a tobacco nicotine demethylase or an RNA molecule that can induce RNA interference (RNAi) operably linked to a functional promoter in plant cells to provide transformed plant cells; and (b) regenerating a plant or plant component from transformed plant cells, in which the antisense or an RNA molecule that can induce RNA interference (RNAi) of the tobacco nicotine demethylase coding sequence is expressed in plant cells or plant component, thereby reducing the expression of tobacco nicotine demethylase in a plant or plant component. Desirably, the transgene encoding an antisense sequence or an RNA molecule that can induce RNA interference (RNAi) of a tobacco nicotine demethylase is constitutively expressed or induciblely expressed, for example in a specific manner. tissue, cell specific or organ specific.

A further aspect of the description describes yet another method for reducing the expression of tobacco nicotine demethylase in a plant or plant component. This method involves the steps of: (a) introducing into a plant cell a transgene that encodes a dominant negative gene product of a tobacco nicotine demethylase operably linked to a functional promoter in the plant cells to provide transformed plant cells; and (b) regenerating a plant or plant component from the transformed plant cells, in which the dominant negative gene product of tobacco nicotine demethylase is expressed in the cells of the plant or plant component, thereby reducing the expression of tobacco nicotine demethylase in a plant or plant component. In particular embodiments of this aspect of the description, the transgene encoding the negative dominant gene product is expressed constitutively or inducibly expressed, for example, in a tissue-specific, cell-specific or organ-specific manner.

A further aspect of the description describes an additional method for reducing the expression or enzymatic activity of tobacco nicotine demethylase in a plant cell. This method involves reducing the level of an endogenous tobacco nicotine demethylase, or its enzymatic activity, in the plant cell. Desirably, the plant cell is from a dicot, a sunflower plant or a species of Nicotiana. In desirable embodiments of this aspect, reducing the level of endogenous tobacco nicotine demethylase involves expressing a transgene encoding an antisense nucleic acid molecule or an RNA molecule that can induce RNA interference (RNAi) of a nicotine demethylase from tobacco in the plant cell, or involves expressing a transgene that encodes a double stranded RNA molecule of a tobacco nicotine demethylase in the plant cell. Desirably, the double stranded RNA is an RNA sequence that corresponds to the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or a fragment Of the same. In a further embodiment, reducing the level of endogenous tobacco nicotine demethylase involves the co-suppression of endogenous tobacco nicotine demethylase in the plant cell or involves expressing a dominant negative gene product in the plant cell. In particular, the dominant negative gene product may include a gene that codes for a mutated form of the amino acid sequence of SEQ ID NO: 2.

In other desirable embodiments of this aspect of the description, the endogenous tobacco nicotine demethylase includes a point mutation in a gene encoding the amino acid sequence of SEQ ID NO: 2. In other desirable embodiments, reducing the level of expression of an endogenous tobacco nicotine demethylase implies a deletion in a gene that encodes a tobacco nicotine demethylase or involves an insertion in a gene that encodes a tobacco nicotine demethylase. Expression reduction can occur at the transcriptional level, at the translational level or at the post-translational level.

A further aspect of the description describes a method for identifying a compound that alters the expression of a tobacco nicotine demethylase in a cell. This method involves the steps of: (a) providing a cell that contains a gene encoding a tobacco nicotine demethylase; (b) apply a candidate compound to the cell; and (c) measuring the expression of the gene encoding tobacco nicotine demethylase, in which an increase or decrease in expression with respect to an untreated control sample is an indication that the compound alters nicotine expression tobacco demethylase

In a desirable embodiment of this method, the gene in part (a) encodes a tobacco nicotine demethylase having an identity of at least 70% with the amino acid sequence of SEQ ID NO: 2. Desirably, the compound decreases or increases the expression of the gene encoding tobacco nicotine demethylase.

In another aspect, the description describes another method for identifying a compound that alters the activity of a tobacco nicotine demethylase in a cell. This method involves the steps of: (a) providing a cell that expresses a gene encoding a tobacco nicotine demethylase; (b) apply a candidate compound to the cell; and (c) measuring the activity of tobacco nicotine demethylase, in which an increase or decrease in activity with respect to an untreated control sample is an indication that the compound alters the activity of tobacco nicotine demethylase. In a desirable embodiment of this aspect of the description, the stage gene

(a) encodes a tobacco nicotine demethylase having an identity of at least 70% with the amino acid sequence of SEQ ID NO: 2. Desirably, the compound decreases or increases the activity of tobacco nicotine demethylase.

A further aspect of the description describes a tobacco plant or cured vegetable component that contains (i) a reduced level of nicotine demethylase or (ii) a nicotine demethylase having an altered enzymatic activity and a reduced amount of a nitrosamine. Desirably, the plant component is a tobacco leaf or tobacco stalk. In a desirable embodiment, the nitrosamine is nornicotine, and the nornicotine content desirably is less than 5 mg / g, 4.5 mg / g, 4.0 mg / g, 3.5 mg / g, 3.0 mg / g, more desirably less than 2.5 mg / g, 2.0 mg / g, 1.5 mg / g, 1.0 mg / g, more desirably less than 750 µg / g, 500 µg / g, 250 µg / g, 100 µg / g, even more desirably less than 75 µg / g, 50 µg / g, 25 µg / g, 10 µg / g, 7.0 µg / g, 5 , 0 µg / g, 4.0 µg / g, and even more desirably less than 2.0 µg / g, 1.0 µg / g, 0.5 µg / g, 0.4 µg / g, 0.2 µg / g, 0.1 µg / g, 0.05 µg / g, or 0.01 µg / g in which the percentage of secondary alkaloids with respect to the total alkaloid content in it it 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 N'-NNN content desirably is less than 5 mg / g, 4.5 mg / g, 4.0 mg / g, 3.5 mg / g, 3.0 mg / g, more desirably less than 2.5 mg / g, 2.0 mg / g, 1.5 mg / g, 1.0 mg / g, more desirably less than 750 µg / g, 500 µg / g, 250 µg / g, 100 µg / g, even more desirably less than 75 µg / g, 50 µg / g, 25 µg / g, 10 µg / g, 7.0 µg / g, 5.0 µg / g, 4.0 µg / g, and even more desirably less than 2.0 µg / g, 1.0 µg / g, 0.5! g / g, 0.4 µg / g, 0.2 µg / g, 0.1 µg / g, 0.05 µg / g or 0.01 µg / g in which the percentage of secondary alkaloids with respect to the total alkaloid content contained 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 additional desirable embodiments of this aspect of the description, the tobacco plant or cured vegetable component is a dark tobacco, Burley tobacco, chimney-cured tobacco, air-cured tobacco or oriental tobacco.

In addition, the tobacco plant or cured vegetable component of the description desirably includes a recombinant nicotine demethylase gene, for example, one containing the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a fragment thereof. Desirably, the expression of an endogenous nicotine demethylase gene in the tobacco plant or cured plant component is silenced.

Another aspect of the description describes a tobacco product that contains a tobacco plant or cured vegetable component that includes (i) reduced expression of a nicotine demethylase or (ii) a nicotine demethylase having altered activity, and a reduced amount of a nitrosamine . Desirably, the tobacco product is smokeless tobacco, dry or wet snuff, chewing tobacco, cigarette, cigar, thin cigar, pipe tobacco or bidis. In particular, the tobacco product of this aspect of the description may contain dark tobacco, ground tobacco

or includes a flavoring component.

The description also describes a method of preparing a tobacco product, for example, a smokeless tobacco product, which contains (i) reduced expression of a nicotine demethylase or (ii) a nicotine demethylase having altered enzymatic activity (for example , reduced), and a reduced amount of a nitrosamine. This method involves providing a tobacco plant or cured vegetable component that contains (i) a reduced level of nicotine demethylase or (ii) a nicotine demethylase having an altered enzymatic activity and a reduced amount of a nitrosamine and preparing the tobacco product to from the tobacco plant or cured vegetable component.

Definitions

"Enzymatic activity" is intended to include but not limited to demethylation, hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S- and O-desalkylations, desulfurization, deamination and reduction of azo, nitro, N-oxide and other chemical groups enzymatically Reagents of this type. Altered enzymatic activity refers to a decrease in the enzymatic activity (for example, of a tobacco nicotine demethylase) by at least 10-20%, preferably at least 25-50% and more preferably at least 55- 95% or higher with respect to the activity of a control enzyme (for example, a nicotine demethylase from a natural-type tobacco plant). The activity of a tobacco nicotine demethylase can be tested using conventional methods in the art, such as measuring the demethylation of radioactive nicotine by microsomes expressed in yeast, as described herein.

The term "nucleic acid" refers to a polymer of deoxyribonucleotides or ribonucleotides in either mono or double stranded, or sense or antisense form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize with nucleic acids. in a manner similar to nucleotides that occur naturally. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof. The terms "operably linked", "in operational combination" and "in operational order" refer to the functional link between a control sequence of nucleic acid expression (such as a promoter, signal sequence or alignment of factor binding sites of transcription) and a second nucleic acid sequence, in which 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 reference to a cell indicates that the cell replicates a heterologous nucleic acid, expresses the nucleic acid or expresses a peptide, heterologous peptide or protein encoded by a heterologous nucleic acid. Recombinant cells can express genes or gene fragments in either a sense or antisense form or an RNA molecule that can induce RNA interference (RNAi) that is not within the native (non-recombinant) form of the cell. Recombinant cells can also express genes that are in the native form of the cell, but in which the genes are modified and reintroduced into the cell by artificial means.

A "structural gene" is that part of a gene that comprises a segment of DNA that encodes a protein, polypeptide or a part thereof, and that excludes, for example, the 5 'sequence that directs the initiation of transcription or UTR in 3 '. The structural gene may alternatively code for a non-translatable product. The structural gene may be one that is normally found in the cell or one that is not normally found in the cell or cellular location in which it was introduced, in which case it is called a "heterologous gene." A heterologous gene can be derived in whole or in part from any source known in the art, including a bacterial episome or genome, eukaryotic, plasmid or nuclear DNA, cDNA, viral DNA or chemically synthesized DNA. A structural gene may contain one or more modifications that could affect biological activity.

or its characteristics, the biological activity or the chemical structure of the expression product, the expression rate or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions and substitutions of one or more nucleotides.

The structural gene may constitute an unbroken coding sequence or may include one or more introns, joined by the appropriate splice junctions. The structural gene can be translatable or non-translatable, including an antisense or an RNA molecule that can induce RNA interference (RNAi). The structural gene may be an amalgam of segments derived from a plurality of sources and a plurality of gene sequences (which occur naturally or synthetically, in which synthetic refers to DNA that is chemically synthesized).

An "exon" as used herein in reference to a nucleic acid sequence refers to a part of the nucleic acid sequence of a gene, in which the exon nucleic acid sequence encodes for at least one amino acid of the gene product. An exon is normally adjacent to a segment of non-coding DNA such as an intron. Desirably, an exon encodes a portion of the amino acid sequence of tobacco nicotine demethylase of SEQ ID NO: 2, such as amino acids 1-313 and / or 314-517 of the sequence of SEQ ID NO: 2.

An "intron" as used herein in reference to a nucleic acid sequence refers to a non-coding region of a gene that is flanked by coding regions. An intron is normally a non-coding region of a gene that is transcribed into an RNA molecule but then cut by RNA splicing during the production of messenger RNA or other functional structural RNA. Desirably, an intron includes the sequence of SEQ ID NO: 5, or a fragment thereof.

A "3" UTR "as used herein in reference to a nucleic acid sequence refers to a non-coding nucleic acid sequence proximal to an exon termination codon. Desirably, a 3 ’UTR includes the sequence of SEQ ID NO: 7, or a fragment thereof.

"Derived from" is used to refer to taken, obtained, received, indicated, replicated or descended from a source (chemical and / or biological). A derivative can be produced by chemical or biological manipulation (including, but not limited to, substitution, addition, insertion, deletion, extraction, isolation, mutation and replication) of the original source.

"Chemically synthesized," in relation to a DNA sequence, means that parts of the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA can be achieved using well established procedures (Caruthers, Methodology of DNA and RNA Sequencing, (1983), Weissman (ed.), Praeger Publishers, New York, chapter 1); Automated chemical synthesis can be performed using one of several commercially available machines.

Optimal sequence alignment can be carried out for comparison, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math 2: 482 (1981), using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by searching for the similarity method of Pearson and Lipman Proc. Natl Acad. Sci. (USA) 85: 2444 (1988), through computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), Or through inspection.

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

The terms "substantial amino acid identity" or "substantial amino acid sequence identity" as applied to amino acid sequences and as used herein indicate a characteristic of a polypeptide, the peptide comprising a sequence having an identity. of sequence of at least 70 percent, preferably an amino acid sequence identity of 80 percent, more preferably an amino acid sequence identity of 90 percent and most preferably a sequence identity of at least 99 to 100 per One hundred as compared to the protein sequence of SEQ ID NOS: 2 and / or 4. Desirably, for a nicotine demethylase, sequence comparison desirably compares a region that follows the cytochrome p450 motif (GXRXCX (G / A) ; SEQ ID NO: 29) with the termination codon of the translated peptide.

The terms "substantial nucleic acid identity" or "substantial nucleic acid sequence identity" as applied to nucleic acid sequences and as used herein indicate a characteristic of a polynucleotide sequence, the polynucleotide comprising a sequence having a sequence identity of at least 50 percent, preferably 60, 65, 70 or 75 percent, more preferably a nucleic acid sequence identity of 81 or 91 percent and most preferably a sequence identity of at least 95, 99 or even 100 percent as compared to SEQ ID NOS: 1, 3, 5, 6, and / or 7. Desirably, for a nicotine demethylase nucleic acid sequence , the comparison desirably compares a sequence encoding a region following the cytochrome p450 motif (GXRXCX (G / A); SEQ ID NO: 29) with the termination codon of the translated peptide.

Another indication that the nucleotide sequences are substantially identical is whether two molecules hybridize to each other under stringent conditions. Rigorous conditions are sequence dependent and will be different in different circumstances. Generally, the stringent conditions are selected to be from about 5 ° C to about 20 ° C, usually from about 10 ° C to about 15 ° C, lower than the thermal melting point (Tm) for the specific sequence at a defined pH and ionic strength. The Tm is the temperature (in defined pH and ionic strength) at which 50% of the target sequence hybridizes with a paired probe. Normally, 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 ° C. For example, in a conventional Southern hybridization procedure, stringent conditions will include an initial wash in 6x SSC at 42 ° C followed by one or more additional washes in 0.2x SSC at a temperature of at least about 55 ° C, usually about 60 ° C , and often about 65 ° C.

Nucleotide sequences are also substantially identical for the purposes of this invention when said nucleotide sequences encode polypeptides and / or proteins that are substantially identical. Therefore, when a nucleic acid sequence encodes essentially for the same polypeptide as a second nucleic acid sequence, the two nucleic acid sequences are substantially identical even if they did not hybridize under stringent conditions due to the degeneracy allowed by the genetic code (see , Darnell et al. (1990) Molecular Cell Biology, second edition Scientific American Books WH Freeman and Company New York for an explanation of codon degeneration and the genetic code). The purity or homogeneity of proteins can be indicated by various means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization after staining. For certain purposes, high resolution may be needed and HPLC or similar means may be used for purification.

By an antibody that "specifically binds" or "specifically recognizes" a tobacco nicotine demethylase means an increase in the affinity of the antibody for a tobacco nicotine demethylase with respect to an equal amount of any other protein. For example, an antibody that specifically binds to a tobacco nicotine demethylase containing the amino acid sequence of SEQ ID NO: 2 desirably has an affinity for its antigen that is at least 2 times, 5 times, 10 times, 30 times or 100 times higher than for an equal amount of any other antigen, including related antigens. The binding of an antibody to an antigen, for example, tobacco nicotine demethylase, can be determined by any of several conventional methods in the art, for example, Western blot analysis, ELISA or joint immunoprecipitation.

As used herein, the term "vector" is used in reference to nucleic acid molecules that transfer segment (s) of DNA to a cell. A vector can act by replicating the DNA and can reproduce independently in a host cell. The term "vehicle" is sometimes used interchangeably with "vector." The term "expression vector" as used herein refers to a recombinant DNA molecule that contains a desired coding sequence and acid sequences. Appropriate nucleic necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for prokaryotic expression usually include a promoter, an operator (optional) and a ribosome binding site, often together with other sequences. Desirably, the promoter includes the sequence of SEQ ID NO: 6, or a fragment thereof that directs transcription. Promoter sequences that have a sequence identity of at least 50%, 60%, 75%, 80%, 90%, 95% or even 99% with the sequence of SEQ ID NO are also desirable: 6 and who direct the transcript. It is known that eukaryotic cells use promoters, enhancers and polyadenylation and termination signals, such as the 3 ’UTR sequence of SEQ ID NO: 7. In some cases, it has been observed that plant expression vectors require the presence of plant-derived introns, such as the intron having the sequence of SEQ ID NO: 5, to have stable expression. As such, the sequence of SEQ ID NO: 5, or any other intron having an appropriate RNA splice junction can be used as described further herein.

For the purpose of regenerating genetically engineered plants complete with roots, a nucleic acid can be inserted into plant cells, for example, by any technique such as inoculation in vivo or by any of the in vitro tissue culture techniques known to produce cells transformed vegetables that can be regenerated to give whole plants. Therefore, for example, insertion into plant cells can be by inoculation in vitro by pathogenic or non-pathogenic A. tumefaciens. Other tissue culture techniques of this type can also be used.

"Plant tissue", "plant component" or "plant cell" includes differentiated and undifferentiated plant tissues, including, but not limited to, roots, buds, leaves, pollen, seeds, tumor tissue and various forms of cells in culture, such as individual cells, protoplasts, embryos and corpus callosum. The plant tissue can be in the plant or in an organ, tissue or cell culture.

"Plant cell" as used herein includes plant cells in the plant and plant cells and protoplasts in culture. "CDNA" or "complementary DNA" generally refers to a single stranded DNA molecule with a nucleotide sequence that is complementary to an unprocessed RNA molecule that contains an intron, or a processed mRNA that lacks introns. The cDNA is formed by the action of the enzyme reverse transcriptase on an RNA template.

"Tobacco" as used herein includes curing in chimney, Virginia, Burley, dark, oriental and other types of plants within the Nicotiana genus. The seed of the genus Nicotiana is readily available commercially in the form of Nicotiana tabacum.

"Articles of manufacture" or "tobacco products" include products such as dry and wet snuff, chewing tobacco, cigarettes, cigars, thin cigars, pipe tobacco, bidis and similar tobacco products.

By "gene silencing" is meant a decrease in the level of gene expression (eg, the 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 higher with respect to the level in a control plant (for example, a natural-type tobacco plant). The reduction of such expression levels can be achieved using conventional methods known in the art including, without limitation, RNA interference, triple-strand interference, ribozymes, homologous recombination, virus-induced gene silencing, antisense and cosuppression technologies, expression of a dominant negative gene product or through the generation of mutated genes using conventional mutagenesis techniques, such as those described herein. The levels of a tobacco demethylase nicotine transcript or polypeptide, or both, are monitored according to any conventional technique including, but not limited to, Northern blotting, RNAse protection or immunoblotting.

By a "tobacco nicotine demethylase" or "nicotine demethylase" as used herein, it is meant a polypeptide that is substantially identical to the sequence of SEQ ID NO: 2. Desirably, a tobacco nicotine demethylase can convert nicotine (C10H14N2, also called 3- (1-methyl-2-pyrrolidinyl) pyridine) into nornicotine (C9H12N2). The activity of a tobacco nicotine demethylase can be tested using conventional methods in the art, such as measuring the demethylation of radioactive nicotine by microsomes expressed in yeast, as described herein.

By a "fragment" or "part" of an amino acid sequence of tobacco nicotine demethylase means at least 20, 15, 30, 50, 75, 100, 250, 300, 400 or 500 contiguous amino acids of the sequence of amino acids of SEQ ID NO: 2. Exemplary desirable fragments are amino acids 1-313 of the sequence of SEQ ID NO: 2 and amino acids 314-517 of the sequence of SEQ ID NO: 2. In addition, with respect to a fragment or part of a tobacco nicotine demethylase nucleic acid sequence, desirable fragments include at least 100, 250, 500, 750, 1000 or 1500 contiguous nucleic acids of the nucleic acid sequence of SEQ ID NO: 1. Exemplary desirable fragments are nucleic acids 1-2009, 2010-2949, 2950-3946, 3947-4562, 4563-6347 and 4731-6347 of the sequence of SEQ ID NO: 1. Other desirable fragments include nucleic acids 1-100, 101-250, 251-500, 501-750 and 751-998 of SEQ ID NO: 5, nucleic acids 1398, 1-1400, 1401-2009, 1840-2009, 1940-2009, 399-1240 and 1241-2009 of SEQ ID NO: 6, and nucleic acids 1100, 101-250, 251-500, 501-750, 751-1000, 1001-1250, 1251-1500 and 1501- 1786 of SEQ ID NO: 7.

By a "substantially pure polypeptide" is meant a tobacco nicotine demethylase that has been separated from most of the naturally occurring components; however, other proteins found in the microsomal fraction associated with a preparation having a nicotine demethylase activity of at least 8.3 pKat / mg protein, 9 pKat / mg protein, 9.5 pKat / mg protein, 10 pKat / mg protein, 10.5 pKat / mg or 10.8 5 pKat / mg protein is also considered to be a substantially pure polypeptide. Typically, the polypeptide is substantially pure when at least 60% by weight of the naturally occurring proteins and organic molecules with which it is naturally associated is free. Preferably, the preparation is at least 75%, more preferably at least 90% and most preferably at least 99%, by weight, a tobacco nicotine demethylase. A tobacco nicotine demethylase can be obtained substantially

10 pure, for example, by extraction from a natural source (for example, a tobacco plant cell); by expression of a recombinant nucleic acid encoding a tobacco nicotine demethylase; or by chemical synthesis of the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis or by HPLC analysis.

By "isolated nucleic acid molecule" is meant a free nucleic acid sequence of the sequences of

15 nucleic acid that naturally flanks the sequence of the nucleic acid molecule in the genome of an organism.

By "transformed cell" is meant a cell into which (or within an ancestor from which) a DNA molecule has been introduced, by means of recombinant DNA techniques, for example, a DNA molecule that encodes for a tobacco nicotine demethylase.

20 As provided herein, the terms "cytochrome p450" and "p450" are used interchangeably.

Brief description of the drawings

Figure 1 is a schematic diagram of the genomic structure of the tobacco nicotine demethylase gene.

Figures 2-1 to 2-3 are the genomic nucleic acid sequence of tobacco nicotine demethylase (SEQ ID 25 NO: 1) and its translation product (SEQ ID NO: 2).

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

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

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

Figure 6 is the 3 ’nucleic acid sequence of the tobacco nicotine demethylase gene (SEQ ID NO: 7).

Detailed description

35 Identification of a genomic clone encoding a tobacco nicotine demethylase

According to the present invention, the RNA was extracted from Nicotiana tissue of converting and non-converting Nicotiana lines. The extracted RNA was then used to create cDNA. The nucleic acid sequences of the present invention were then generated using two strategies.

In the first strategy, RNA enriched in poly A was extracted from plant tissue and cDNA was prepared by PCR

40 with reverse transcription. Then, the single stranded cDNA was used to create specific PCR populations of p450 using degenerated primers plus an inverse oligo d (T) primer. The primer design was based on the highly conserved motifs of cytochrome p450 gene sequences from other plants. Examples of specific degenerate primers are set forth in Figure 1 of patent application publications US 2004/0103449 A1 and US 2004/0111759 A1. The sequence of plasmid fragments which

45 contained inserts of appropriate size. These size inserts typically range from about 300 to about 800 nucleotides depending on which primers were used.

In a second strategy, a cDNA library was initially constructed. The cDNA in the plasmids was used to create specific PCR populations of p450 using degenerate primers plus a T7 primer in the plasmid as a reverse primer. As in the first strategy, the sequence of plasmid fragments containing inserts of appropriate size was further analyzed.

Starting lines of Nicotiana plants that are known to produce high levels of nornicotine (converters) and plant lines that have low levels of nornicotine can be used as starting materials. Then, the leaves of the plants can be removed and treated with ethylene to activate the enzymatic activities of p450 defined herein. Total RNA is extracted using techniques known in the art. Then, cDNA fragments can be generated using PCR (RT-PCR) with the oligo d (T) primer. Then, the cDNA library can be constructed as described more fully in the examples herein.

The conserved region of p450 type enzymes was used as a template for degenerate primers. Using degenerate primers, specific bands of p450 were amplified by PCR. Indicative bands were identified for enzymes similar to p450 by DNA sequencing. PCR fragments were characterized using BLAST search, alignment or other tools to identify appropriate candidates.

Sequence information from identified fragments was used to develop the PCR primers. These primers, in combination with plasmid primers in the cDNA library, were used to clone full-length p450 genes. Large-scale inverse Southern type analysis was performed to examine the differential expression of all clones of fragments obtained and in some cases full-length clones. In this aspect of the invention, these large-scale inverse Southern-type assays can be performed using labeled total cDNAs from different tissues as a probe to hybridize with cloned DNA fragments in order to select all cloned inserts. Non-radioactive and radioactive Northern blotting assays (P32) were also used to characterize the cloned p450 fragments and full length clones.

Peptide specific antibodies were prepared by deriving their amino acid sequence and selecting peptide regions that were antigenic and unique with respect to other clones. Rabbit antibodies against synthetic peptides conjugated to a carrier protein were prepared. Western blot analysis or other immunological methods were performed on plant tissue using these antibodies. In addition, peptide specific antibodies were prepared for several full length clones by deriving their amino acid sequence and selecting peptide regions that were potentially antigenic and were unique with respect to other clones. Rabbit antibodies against synthetic peptides conjugated to a carrier protein were prepared. Western blot analysis was performed using these antibodies.

Regulation for decreased nicotine demethylase tobacco

Plants are generated that have a decreased expression of a tobacco nicotine demethylase according to conventional gene silencing methods. (For reviews, see Arndt and Rank, Genome 40: 785-797, 1997; Turner and Schuch, Journal of Chemical Technology and Biotechnology 75: 869-882, 2000; and Klink and Wolniak, Journal of Plant Growth Regulation 19 (4) : 371-384, 2000.) In particular, the tobacco nicotine demethylase gene promoter (for example, SEQ ID NO: 6), the structural gene (SEQ ID NO: 3), the intron (SEQ ID NO: 5 ) or the 3 'UTR (SEQ ID NO: 7) or the entire genomic clone (SEQ ID NO: 1) can be used to alter tobacco phenotypes or tobacco metabolites, for example, nornicotine in any Nicotiana species. Decreased expression of a tobacco nicotine demethylase gene can be achieved using, for example, RNA interference (RNAi) (Smith et al., Nature 407: 319-320, 2000; Fire et al., Nature 391: 306 -311, 1998; Waterhouse et al., PNAS 95: 1395913964, 1998; Stalberg et al., Plant Molecular Biology 23: 671-683, 1993; Brignetti et al., EMBO J. 17: 6739-6746, 1998; Allen et al., Nature Biotechnology 22: 1559-1566, 2004); 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 transferring an endogenous plant gene in the sense orientation (Jorgensen et al., Plant Mol Biol 31: 957-973, 1996); antisense gene expression; homologous recombination (Ohl et al., Homologous Recombination and Gene Silencing in Plants (Kluwer, Dordrecht, The Netherlands, 1994); Cre / lox systems (Qin et al., PNAS 91: 1706-1710, 1994; Koshinsky et al., The Plant Journal 23: 715-722, 2000; Chou, et al., Plant and Animal Genome VII Conference Abstracts. San Diego, CA, January 17-21, 1999); gene entrapment and T-DNA labeling (Burns et al., Genes Dev. 8: 1087-1105, 1994; Spradling, et al., PNAS 92: 10824-10830, 1995; Skarnes et al., Bio / Technology 8, 827-831, 1990; Sundaresan, et al. , Genes Dev. 9: 1797-1810, 1995); and any of the other possible gene silencing systems that are available in the areas of science that result in regulation by decreased expression of a tobacco nicotine demethylase or a reduction in its enzymatic activity. Example methods are described in more detail below.

RNA interference

RNA interference ("RNAi") is a generally applicable process for inducing potent and specific posttranslational gene silencing in many organisms including plants (see, for example, Bosher et al., Nat. Cell Biol. 2: E31-36, 2000 ; and Tavernarakis et al., Nat. Genetics 24: 180-183, 2000). RNAi involves the introduction of RNA partially or completely double-stranded in the cell or in the extracellular environment. The inhibition is specific because a nucleotide sequence of a part of the target gene (for example, a tobacco nicotine demethylase) is chosen to produce inhibitory RNA. The chosen part generally encompasses exons of the target gene, but the chosen part may also include untranslated regions (UTR), as well as introns (for example, the sequences of SEQ ID NO: 5 or 7).

For example, to construct transformation vectors that produce RNA that can form duplex, two nucleic acid sequences of tobacco nicotine demethylase, one in the sense orientation and the other in the antisense, can be operably linked, and placed under control. of a strong viral promoter, such as CaMV 35S or the isolated promoter of Cassava brown ray virus (CBSV). However, the use of the endogenous promoter, such as the tobacco nicotine demethylase promoter having the sequence of SEQ ID NO: 6, or a fragment thereof that directs transcription, may also be desirable. The length of the tobacco nicotine demethylase nucleic acid sequences included in such a construct is desirably at least 25 nucleotides, but may encompass a sequence that includes up to the full-length tobacco nicotine demethylase gene.

Constructs that produce RNA that can form duplex can be introduced into the genome of a plant, such as a tobacco plant, by Agrobacterium-mediated transformation (Chuang et al., Proc. Natl. Acad. Sci. USA 97: 4985-4990, 2000), producing inheritable and specific genetic interference in a tobacco nicotine demethylase. Double stranded RNA can also be introduced directly into the cell (i.e. intracellularly) or introduced extracellularly, for example, by bathing a seed, seedling or plant in a solution containing the double stranded RNA.

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

The RNA used in the RNAi may comprise one or more strands of polymerized ribonucleotide; it can include modifications in either the main phosphate-sugar structure or the nucleoside. The double-stranded structure can be formed by a single strand of self-complementary RNA or by two strands of complementary RNA and the duplex formation of RNA can be initiated either inside or outside the cell. The RNA can be introduced in an amount that allows the supply of at least one copy per cell. However, higher doses (for example, at least 5, 10, 100, 500 or 1000 copies per cell) of double stranded material may provide a more effective inhibition. The inhibition is sequence specific because the nucleotide sequences corresponding to the duplex region of the RNA are selected as a target for genetic inhibition. RNA containing a nucleotide sequence identical to a part of the target gene is preferred for inhibition. RNA sequences with individual insertions, deletions and point mutations with respect to the target sequence may also be effective for inhibition. Therefore, sequence identity can be optimized by alignment algorithms known in the art and by calculating the percentage of differences between nucleotide sequences. Alternatively, the RNA duplex region can be functionally defined as a nucleotide sequence that can hybridize with a portion of the target gene transcript.

In addition, the RNA used for RNAi can be synthesized either in vivo or in vitro. For example, endogenous RNA polymerase in the cell can mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro. For transcription from an in vivo transgene or an expression construct, a regulatory region can be used to transcribe the strand of RNA (or strands).

Triple strand interference

The gene expression of endogenous tobacco nicotine demethylase can also be regulated by decrease by selecting target deoxyribonucleotide sequences complementary to the regulatory region of a tobacco nicotine demethylase gene (e.g., enhancer or promoter regions) to form triple helix structures that they prevent the transcription of the tobacco nicotine demethylase gene into target cells. (See generally, Helene, Anticancer Drug Des. 6: 569-584, 1991; Helene et al., Ann. NY Acad. Sci. 660: 27-36, 1992; and Maher, Bioassays 14: 807-815, 1992 .)

The nucleic acid molecules used in the triple helix formation for transcription inhibition are preferably single stranded and are composed of deoxyribonucleotides. The base composition of these oligonucleotides should promote triple helix formation by means of Hoogsteen base pairing rules, which generally require that considerable sections of either purines or pyrimidines be present in a strand of a duplex. The nucleotide sequences can be based on pyrimidines, which will result in TAT and CGC triplets through the three associated strands of the resulting triple helix. Pyrimidine-rich molecules provide base complementarity with a purine-rich region of an individual strand of the duplex in an orientation parallel to that strand. In addition, nucleic acid molecules that are rich in purines can be chosen, for example, that contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of Purine residues are located in an individual strand of the selected duplex as a target, resulting in CGC triplets across the three strands in the triplex.

Alternatively, the possible sequences that can be selected as the target for triple helix formation can be augmented by creating a "sense-changing" nucleic acid molecule. The sense change molecules are synthesized in an alternating 5'-3 ', 3'-5' manner, so that their bases appear first with a strand of one duplex and then with the other, eliminating the need for a considerable section of

either purines or pyrimidines in a strand of a duplex.

Ribozymes

Ribozymes are RNA molecules that act as enzymes and can be engineered to cleave other RNA molecules. A ribozyme can be designed to specifically mate with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. Ribozyme itself is not consumed in this process and can act catalytically to cleave multiple copies of mRNA target molecules. Accordingly, ribozymes can also be used as means to regulate by decreasing the expression of a tobacco nicotine demethylase. The design and use of specific ribozymes of target RNA is described in Haseloff et al. (Nature 334: 585-591, 1988). Preferably, the ribozyme includes at least about 20 continuous nucleotides complementary to the target sequence (eg, a tobacco nicotine demethylase) on each side of the active ribozyme site.

In addition, ribozyme sequences can also be included within an antisense RNA to confer RNA cleavage activity to the antisense RNA and thereby increase the efficacy of the antisense construct.

Homologous Recombination

Gene replacement technology is another desirable method for regulating by decreasing the expression of a given gene, for example, a tobacco nicotine demethylase. Gene replacement technology is based on homologous recombination (see, Schnable et al., Curr. Opinions Plant Biol. 1: 123-129, 1998). The nucleic acid sequence of the enzyme of interest such as a tobacco nicotine demethylase can be manipulated by mutagenesis (for example, insertions, deletions, duplications or substitutions) to decrease enzyme function. The altered sequence can then be introduced into the genome to replace the existing gene, for example, wild-type, by homologous recombination (Puchta et al., Proc. Natl. Acad. Sci. USA 93: 5055-5060, 1996; and Kempin et al., Nature 389: 802-803, 1997).

Cosuppression

An additional desirable method of silencing gene expression is cosuppression (also called sense suppression). It has been shown that this technique, which involves the introduction of a nucleic acid configured in the sense orientation, effectively blocks the transcription of target genes (see, for example, Napoli et al., Plant Cell, 2: 279-289, 1990 and Jorgensen et al., U.S. Patent No. 5,034,323).

Generally, sense suppression involves transcription of the introduced sequence. However, cosuppression can also occur when the sequence introduced does not contain any coding sequence per se, but only an intron (for example, the sequence of SEQ ID NO: 5) or untranslated sequences such as the sequence of SEQ ID NO: 7 or other sequences of this type substantially identical to sequences present in the primary transcript of the endogenous gene to be repressed. The sequence introduced will generally be substantially identical to the endogenous gene selected as a target for repression. Such an identity is normally greater than about 50%, but higher identities (for example, 80% or even 95%) are preferred because they result in more effective repression. The effect of cosuppression can also be applied to other proteins within a similar family of genes that show substantial homology or homology. Segments of a plant gene can be used directly, for example, to inhibit the expression of homologous genes in different plant species.

In the sense suppression, the sequence introduced, requires less than absolute identity, does not need to be full length, with respect to either the primary transcription product or fully processed mRNA. A higher degree of sequence identity in a shorter full length sequence compensates for a longer sequence of lower identity. In addition, the sequence introduced does not need to have the same pattern of introns or exons, and the identity of non-coding segments can be equally effective. Sequences of at least 50 base pairs are preferred, with more introduced sequences of greater length being preferred (see, for example, the methods described by Jorgensen et al., U.S. Patent No. 5,034,323).

Antisense suppression

In antisense technology, a nucleic acid segment of the desired plant gene, such as that found in SEQ ID NOS: 1, 3, 5, 6 or 7, is cloned and operably linked to a control region of the expression so that the antisense strand of the RNA is synthesized. Then, the construct is transformed into plants and the antisense strand of RNA is produced. In plant cells, antisense RNA has been shown to inhibit gene expression.

Generally, the segment of nucleic acid to be introduced in antisense suppression is substantially identical to at least a part of the endogenous gene or genes to be repressed, but does not need to be identical. The tobacco nicotine demethylase nucleic acid sequences disclosed herein may be included in vectors designed so that the inhibitory effect is applied to other proteins within a family of genes that show substantial homology or homology to the gene. Diana. Segments of a plant gene can be used, for example, directly to inhibit the expression of homologous genes in different tobacco varieties.

The sequence introduced also does not need to be full length with respect to either the primary transcription product or the fully processed mRNA. Generally, a higher homology can be used to compensate for the use of a shorter sequence. In addition, the sequence introduced does not need to have the same pattern of introns or exons, and the homology of non-coding segments will be equally effective. In general, such an antisense sequence will usually have at least 15 base pairs, preferably approximately 15-200 base pairs and more preferably 200-2,000 base pairs in length or greater. The antisense sequence may be complementary to all or a part of the gene to be deleted (eg, a tobacco nicotine demethylase promoter (SEQ ID NO: 6), exon, intron (SEQ ID NO: 5) or UTR (SEQ ID NO: 7) and, as those skilled in the art appreciate, the particular site or sites to which the antisense sequence binds as well as the length of the antisense sequence will oscillate, depending on the degree of inhibition desired and the uniqueness of the antisense sequence A transcriptional construct that expresses a negative regulatory antisense nucleotide sequence of plants includes, in the transcription direction, a promoter, the sequence encoding the antisense RNA in the sense strand and a region of transcription termination. antisense sequences can be constructed and expressed as described, for example, in van der Krol et al. (Gene 72: 4550, 1988); Rodermel et al. (Cell 55: 673-681, 1988); Mol et al. ( FEBS Lett. 26 8: 427-430, 1990); Weigel and Nilsson (Nature 377: 495-500, 1995); Cheung et al., (Cell 82: 383-393, 1995); and Shewmaker et al. (U.S. Patent No. 5,107,065).

Negative dominants

Transgenic plants expressing a transgene that codes for a dominant negative gene product of a tobacco nicotine demethylase in artificial environments or in the field can be tested to demonstrate that the transgene confers regulation by decreasing a tobacco nicotine demethylase in the transgenic plant . Dominant negative transgenes are constructed according to methods known in the art. Normally, a negative dominant gene encodes a mutant negative regulatory polypeptide of a tobacco nicotine demethylase which, when overexpressed, alters the activity of the wild-type enzyme.

Mutants

Plants that have enzymatic activity or decreased expression of a tobacco nicotine demethylase can also be generated using conventional mutagenesis methodologies. Such methods of mutagenesis include, without limitation, treatment of seeds with ethyl methylsulfate (Hildering and Verkerk, in, The use of induced mutations in plant breeding. Pergamon press, pages 317-320, 1965) or UV radiation, X-rays and irradiation with fast neutrons (see, for example, Verkerk, Neth. J. Agric. Sci. 19: 197-203, 1971; and Poehlman, Breeding Field Crops, Van Nostrand Reinhold, New York (3.sup.rd ed), 1987 ), use of transposons (Fedoroff et al., 1984; U.S. Patent No. 4,732,856 and U.S. Patent No. 5,013,658), as well as T-DNA insertion methodologies (Hoekema et al., 1983; patent U.S. 5,149,645). Types of mutations that may be present in a tobacco nicotine demethylase gene include, for example, point mutations, deletions, insertions, duplications and inversions. Such mutations are desirably present in the coding region of a tobacco nicotine demethylase gene; however, mutations in the promoter region, and intron, or an untranslated region of a tobacco nicotine demethylase gene may also be desirable.

For example, insertional T-DNA mutagenesis can be used to generate insertional mutations in a tobacco nicotine demethylase gene to regulate by decreasing gene expression. Theoretically, approximately 100,000 independent T-DNA insertions are required for a 95% chance of obtaining an insertion in any given gene (McKinnet, Plant J. 8: 613-622, 1995; and Forsthoefel et al., Aust. J. Plant Physiol. 19: 353-366, 1992). Plant lines labeled with T-DNA can be selected using polymerase chain reaction analysis (PCR). For example, a primer for one end of the T-DNA can be designed and another primer can be designed for the gene of interest and both primers can be used in the PCR analysis. If a PCR product is not obtained, then there is no insertion in the gene of interest. However, if a PCR product is obtained, then there is an insertion in the gene of interest.

The expression of a mutated tobacco nicotine demethylase can be evaluated according to conventional procedures (for example, those described herein) and, optionally, can be compared with the expression of the non-mutated enzyme. When compared to non-mutated plants, mutated plants that have decreased expression of a gene encoding a tobacco nicotine demethylase are desirable embodiments of the present invention.

Plant promoters

An example of a plant promoter useful according to the invention is the nicotine demethylase promoter having the sequence of SEQ ID NO: 6, or a fragment thereof that directs transcription. Other desirable promoter is a caulimovirus promoter, for example, a 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 not dependent on virally encoded proteins. CaMV is a source for both 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. In addition, the activity of this promoter can be further increased (ie, 2-10 times) by duplication of the CaMV 35S promoter.

Other useful plant promoters include, without limitation, the nopaline synthase promoter (NOS), the octopine synthase promoter, the scrofular mosaic virus (FMV) promoter, the rice actin promoter and the ubiquitin promoter system.

Exemplary monocotyledoneone promoters include, without limitation, the Commelina yellow spotted virus promoter, the sugarcane badnavirus promoter, the rice tungro bacilliform virus promoter, the stripe virus element of corn and the promoter of the wheat dwarf virus.

For certain applications, it may be desirable to produce a tobacco nicotine demethylase, such as a negative dominant mutant tobacco nicotine demethylase, in an appropriate tissue, at an appropriate level, or at a time of appropriate development. To this end, it has been shown that a collection of gene promoters, each with its own distinct characteristics incorporated into its regulatory sequences, are regulated in response to inducible signals such as the environment, hormones and / or developmental signals. These include, without limitation, gene promoters that are responsible for heat-regulated gene expression, light-regulated gene expression (e.g., the pea rbcS-3A gene; the corn rbcS promoter; the binding protein gene a chlorophyll a / b found in the pea; or the Arabssu promoter), the hormone-regulated gene expression (for example, the abscisic acid-sensitive (ABA) sequences of the wheat Em gene; the ABA-inducible HVA1 and HVA22 promoters, and rd29A from barley and Arabidopsis; and wound-induced gene expression (e.g., from wunI), organ-specific gene expression (e.g., from the tuber-specific storage protein gene; the 23 KDa zein gene of the corn described by; or the French pea 1-phaseoline gene), or pathogen-inducible promoters (e.g., PR-1, prp-1, or 1-1,3-glucanase promoters, the wirla fungus-inducible promoter of wheat, and the promoter it is inducible by nematodes, TobRB7-5A and Hmg-1, of tobacco and parsley, respectively).

Plant Expression Vectors

Normally, plant expression vectors include (1) a cloned plant gene (e.g., a tobacco nicotine demethylase gene) under the transcriptional control of 5 'and 3' regulatory sequences (e.g., the nicotine promoter Tobacco demethylase (SEQ ID NO: 6) and the 3 'UTR regions (SEQ ID NO: 7)) and (2) a dominant selectable marker. Such plant expression vectors may also contain; if desired, a promoter regulatory region (for example, one that confers inducible or constitutive expression, tissue or cell specific, regulated by development or by the environment, induced by wounds or pathogens), a site of initiation of the transcription initiation, a ribosome binding site, an RNA processing signal, a transcription termination site and / or a polyadenylation signal.

Plant expression vectors may also optionally include RNA processing signals, for example, introns, which have been shown to be important for the synthesis and accumulation of effective RNA. The location of the RNA splicing sequences can dramatically influence the level of transgenic expression in plants. In view of this fact, an intron can be placed in the 5 ’or 3’ sense of a tobacco nicotine demethylase coding sequence in the transgene to alter gene expression levels.

In addition to the 5 'regulatory control sequences mentioned above, expression vectors may also include regulatory control regions that are generally present in the 3' regions of plant genes. For example, the 3 ’terminator region (for example, the sequence of SEQ ID NO: 7) can be included in the expression vector to increase mRNA stability. A terminator region of this type may be derived from the terminator region PI-II of the potato. In addition, other commonly used terminators are derived from octopine or nopaline synthase signals.

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

The effective use of selectable markers is facilitated by a determination of the susceptibility of a plant cell to a particular selectable agent and a determination of the concentration of this agent that effectively destroys most, if not all, of the transformed cells. Some useful concentrations of antibiotics for tobacco transformation include, for example, 20-100 µg / ml (kanamycin), 20-50 µg / ml (hygromycin) or 5-10 µg / ml (bleomycin). A useful strategy for the selection of transformants for herbicide resistance is described, for example, by Vasil (Cell Culture and Somatic Cell Genetics of Plants, vol. I, II, III Laboratory Procedures and Their Applications Academic Press, New York, 1984 ).

In addition to a selectable marker, it may be desirable to use an indicator gene. In some cases, an indicator gene can be used without a selectable marker. Indicator genes are genes that are not normally present or expressed in the recipient's tissue or organism. The reporter gene normally encodes a protein that provides some phenotypic change or enzymatic property. Examples of such genes are provided in Weising et al. (Ann. Rev. Genetics 22: 421, 1988). Preferred indicator genes include without limitation the glucuronidase gene (GUS) and the GFP genes.

After the construction of the plant expression vector, several conventional methods are available for the introduction of the vector into a plant host, thereby generating a transgenic plant. These methods include (1) Agrobacterium-mediated transformation (A. tumefaciens or A. rhizogenes) (see, for example, Lichtenstein and Fuller in: Genetic Engineering, vol. 6, PWJ Rigby, ed, London, Academic Press, 1987; and Lichtenstein, CP, and Draper, J, In: DNA Cloning, vol. II, DM Glover, ed, Oxford, IRI Press, 1985; U.S. Patent Nos. 4,693,976, 4,762,785, 4,940,838, 5,004,863 , 5,104,310, 5,149,645, 5,159,135, 5,177,010, 5,231,019, 5,463,174, 5,469,976 and 5,464,763; and European patent applications numbers 0131624B1, 0159418B1, 0120516, 0176112, 0116718, 0267159, 0290799, 0292435, 0320500, 0604622 and 0627752), (2) the particle delivery system (see, for example, U.S. Patent Nos. 4,945,050 and 5,141,131), (3) microinjection protocols, (4 ) procedures with polyethylene glycol (PEG), (5) liposome-mediated DNA uptake, (6) electroporation protocols (see, for example, WO 87/06614, WO 92 / 09696 and WO 93/21335; and U.S. Patent Nos. 5,384,253 and 5,472,869, (7) the vortex agitation method or (8) the so-called "monocrystalline short fibers" methodology (see, for example, Coffee et al., U.S. Patent Numbers 5,302,523 and 5,464,765). The type of plant tissue that can be transformed with an expression vector includes embryonic tissue, corpus callosum type I and II, hypocotyls, meristem and the like.

Once introduced into the plant tissue, the expression of the structural gene can be tested by any means known in the art, and the expression can be measured as transcribed mRNA, synthesized protein or the amount of gene silencing that occurs as determined by metabolite monitoring by means of chemical analysis of secondary alkaloids in tobacco (as described herein; see also U.S. Patent No. 5,583,021). Techniques are known for in vitro culture of plant tissue, and in several cases, for regeneration to give whole plants (see, for example, U.S. Patent Nos. 5,595,733 and 5,766,900). Those skilled in the art are aware of methods for transferring the introduced expression complex to commercially useful cultivars.

Once plant cells expressing the desired level 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. Then, the regenerated plants are reproduced by conventional means and the introduced genes can be transferred to other strains and cultivars by conventional plant cultivation techniques.

Transgenic tobacco plants can incorporate a nucleic acid from any part of the genomic gene in different orientations for either decrease regulation, for example, antisense orientation or in a way to induce RNAi, or overexpression, for example, sense orientation. Overexpression of the nucleic acid sequence encoding the complete amino acid sequence or a functional part thereof of a full-length tobacco nicotine demethylase gene is desirable to increase the expression of nicotine demethylase within the Nicotiana lines. .

Determination of the transcriptional or translational levels of a tobacco nicotine demethylase

Tobacco nicotine demethylase expression can be measured, for example, by conventional Nothern-type transfer analysis (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, NY, (1989)) using a tobacco nicotine demethylase (or cDNA fragment) as a hybridization probe. The determination of RNA expression levels can also be aided by reverse transcription PCR (rtPCR), including quantitative rtPCR (see, for example, Kawasaki et al., In PCR Technology: Principles and Applications of DNA amplification (HA Erlich, Ed .) Stockton Press (1989); Wang et al. In PCR Protocols: A Guide to Methods and Applications (MA Innis, et al., Eds.) Academic Press (1990); and Freeman et al., Biotechniques 26: 112- 122 and 124-125, 1999). Additional well-known techniques for determining the expression of a tobacco nicotine demethylase gene include in situ hybridization, and fluorescent in situ hybridization (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, (2001)). The above conventional techniques are also useful for comparing the level of expression between plants, for example, between a plant that has a mutation in a tobacco nicotine demethylase gene and a control plant.

If desired, the expression of a tobacco nicotine demethylase gene can be measured at the level of tobacco nicotine demethylase protein production using the same general approach and conventional protein analysis techniques that include Bradford assays, spectrophotometric assays and techniques. of immunological detection, such as Western blotting or immunoprecipitation with a tobacco-specific nicotine demethylase antibody (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, NY, (1989)).

Identification of modulators of a tobacco nicotine demethylase

The isolation of a tobacco nicotine demethylase cDNA also facilitates the identification of molecules that increase or decrease the expression of a tobacco nicotine demethylase. According to one approach, candidate molecules are added at varying concentrations to a cell culture medium (for example, prokaryotic cells such as E. coli or eukaryotic cells such as yeast, mammalian, insect or plant cells) expressing an mRNA of tobacco nicotine demethylase. Then, the expression of tobacco nicotine demethylase in the presence and absence of a candidate molecule is measured using conventional methods such as those set forth herein.

The candidate modulators can be purified (or substantially purified) molecules or they can be a component of a mixture of compounds. In a mixed compound test, the expression of tobacco nicotine demethylase is tested against progressively smaller subgroups of the candidate compound set (for example, produced by conventional purification techniques, for example, HPLC) until it is demonstrated that a single compound or a mixture of minimal compounds alters the gene expression of tobacco nicotine demethylase. A molecule that promotes a decrease in the expression of tobacco nicotine demethylase is considered particularly useful. Modulators found to be effective at the level of the expression or activity of tobacco nicotine demethylase can be confirmed as useful in the plant.

For agricultural uses, the molecules, compounds or agents identified using the methods disclosed herein can be used as chemicals applied as aerosols or powders on plant foliage. Molecules, compounds or agents can also be applied to plants in combination with another molecule that produces some benefit to the plant.

Uses of the nicotine demethylase gene promoter and untranslated regions

The promoter region of the nicotine demethylase gene described herein is inducible with ethylene or is related to the senescence of the plant. Therefore, this promoter could be used to direct the expression of any desirable gene product to improve crop quality or enhance specific traits. Since the tobacco nicotine demethylase promoter (for example, SEQ ID NO: 6) is inducible and is expressed during a particular period of the plant's life cycle, constructs containing this promoter can be introduced into the plant to express genes uniquely involved in the biosynthesis of flavor and aroma products that result from secondary metabolites. Examples of such compounds are compounds in the terpenoid pathway, other alkaloids, plant hormones, flavonoids or sugar containing moieties. A promoter of

5 tobacco nicotine demethylase to increase or modify the expression of structural carbohydrates or proteins that affect the end-use properties. In addition, a tobacco nicotine demethylase promoter could also be combined with heterologous genes including genes involved in the biosynthesis of nutritional products, pharmaceutical agents or industrial materials. The promoter can be used to direct regulation by decreasing endogenous genes in tobacco including nicotine demethylase or other genes involved in alkaloid biosynthesis and in other routes.

In addition, the promoter region (eg, SEQ ID NO: 6) of a tobacco nicotine demethylase gene or the UTR in 3 '(for example, SEQ ID NO: 7) of a tobacco nicotine demethylase gene can also be used in any site-directed gene silencing method such as T-DNA tagging, gene trapping and homologous recombination to alter the expression pattern of a target gene, as described herein.

15 document. Promoter motifs, which can easily be identified in a promoter sequence, such as the sequence of SEQ ID NO: 6 using conventional methods in the art, can also be used to identify factors that are associated with or regulate the expression of a tobacco nicotine demethylase. Desirably, a tobacco nicotine demethylase promoter region or other transcription regulatory region is used to alter chemical properties such as nornicotine content and nitrosamine levels in a plant.

In addition, any part of a tobacco nicotine demethylase gene can be used as a genetic marker to isolate related genes, regulatory or promoter regions, or to select the demethylase gene in another tobacco or Nicotiana species.

products

Tobacco products that have a reduced amount of nitrosamine content are prepared using any

Material of the tobacco plant described herein according to conventional methods known in the art. In one embodiment, tobacco products are prepared using plant material obtained from a genetically modified cured tobacco having a reduced amount of nornicotine or NNN less than about 5 mg / g, 4.5 mg / g, 4.0 mg / g , 3.5 mg / g, 3.0 mg / g, 2.5 mg / g, 2.0 mg / g, 1.5 mg / g, 1.0 mg / g, 750 µg / g, 500 ! g / g, 250! g / g, 100! g / g, 75! g / g, 50! g / g, 25! g / g, 10! g / g, 7.0! g / g, 5 , 0! G / g, 4.0! G / g, 2.0! G / g, 1.0! G / g, 0.5! G / g, 0.4! G / g, 0.2 ! g / g, 0.1! g / g, 0.05! g / g, or 0.01! g / g in which the percentages of secondary alkaloids with respect to the total alkaloid content contained therein is lower at 90%, 70%, 50%, 30%, 10%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.75 %, 0.5%, 0.25% or 0.1%. That is, cured tobacco is prepared from a genetically modified tobacco plant. The phrase "a reduced amount" is intended to refer to an amount of nornicotine or NNN or both in a tobacco plant, tobacco or a transgenic tobacco product that

35 is lower than what would be found in a tobacco, tobacco or tobacco product that is produced naturally from the same variety of tobacco, processed in the same way, which did not become transgenic material for nornicotine or NNN reduced Therefore, in some contexts, a naturally occurring tobacco of the same variety that has been processed in the same way is used as a control by which to measure whether a reduction of nornicotine or NNN has been obtained by the methods described in This document. Nornicotine and NNN levels are measured according to methods well known in the art of tobacco.

The following examples illustrate methods for carrying out the invention and should be understood to be illustrative of, but not limited to, the scope of the invention defined in the appended claims.

Example 1

Plant tissue development and ethylene treatment

45 Plant growth

Plants were planted in pots and grown in a greenhouse for 4 weeks. The 4-week-old seedlings were transplanted into individual pots and grown in the greenhouse for 2 months. Plants were irrigated 2 times a day with water containing 150 ppm of NPK fertilizer during growth. The expanded green leaves of the plants were detached to perform the ethylene treatment described below.

78379 cell line

The 78379 tobacco line was used, which is a Burley tobacco line ceded by the University of Kentucky as a source of plant material. One hundred plants were grown as a standard in the growing tobacco technique, transplanted and labeled with a distinctive number (1-100). Fertilization and field treatment were performed as recommended.

Three quarters of the 100 plants converted between 20 and 100% of the nicotine into nornicotine. A quarter of the 100 plants converted less than 5% of nicotine into nornicotine. Plant number 87 had the lowest conversion (2%)

5 while plant number 21 had a 100% conversion. Plants that converted less than 3% were classified as non-converters. Self-pollinated seeds of plant number 87 and plant number 21 were prepared, as well as cross seeds (21 x 87 and 87 x 21) to study genetic and phenotypic differences. The plants of the 21 self-fertilized were converters, and 99% of the self-fertilized of the 87 were non-converters. The other 1% of the 87 plants showed low conversion (5-15%). The reciprocal crossing plants were all converters.

4407 cell line

The Nicotiana line 4407, which is a Burley line, was used as a source of plant material. Uniform and representative plants were selected and labeled (100). Of the 100 plants, 97 were non-converters and three were converters. Plant number 56 had the lowest conversion amount (1.2%) and plant number 58 had the

15 highest level of conversion (96%). Self-pollinated seeds and cross seeds were prepared with these two plants.

Plants of the self-fertilized 58 were segregated with a converter ratio with respect to non-converter 3: 1. Plants 58-33 and 58-25 were identified as homozygous converting and non-converting plant lines, respectively. The stable conversion of 58-33 was confirmed by analysis of its progeny.

PBLB01 cell line

PBLB01 is a Burley line developed by ProfiGen, Inc. and was used as a source of plant material. The converter plant was selected from the foundation seeds of PBLB01.

Ethylene treatment procedure

Green leaves were detached from plants grown in a 2-3 month greenhouse and sprayed with

25% ethylene solution (Prep brand Ethephon (Rhone-Poulenc)). Each powdered sheet was hung on a curing rack equipped with a humidifier and covered with plastic. During the treatment, the sample sheets were periodically sprayed with the ethylene solution. Approximately 24-48 hours after treatment with ethylene, the leaves were collected for RNA extraction. Another subsample was taken for the analysis of metabolic constituents to determine the concentration of leaf metabolites and more specific constituents of interest such as a variety of alkaloids.

As an example, the alkaloid analysis can be performed as follows. Samples (0.1 g) were stirred at 150 rpm with 0.5 ml of 2 N NaOH and a 5 ml extraction solution containing quinoline as internal standard and methyl t-butyl ether. The samples were analyzed in an HP 6890 GC apparatus equipped with an FID detector. A temperature of 250 ° C was used for the detector and the injector. An HP column (30 m-0.32 nm-1 mm) consisting of molten silica was used

35 crosslinked with 5% phenol and 95% methylsilicone at a temperature gradient of 110-185 ° C at 10 ° C per minute. The column was operated at 100 ° C with a flow rate of 1.7 cm3min-1 with a distribution ratio of 40: 1 with a 2: 1 injection volume using helium as a carrier gas.

Example 2

RNA isolation

For RNA extractions, intermediate leaves of plants grown in a two month old greenhouse were treated with ethylene as described above. Samples of 0 and 24-48 hours were used for RNA extraction. In some cases, leaf samples were taken in the senescence process of the plants 10 days after the removal of the floral chapter. These samples were also used for extraction. Total RNA was isolated using the Rneasy Plant Mini® kit (Qiagen, Inc., Valencia, California) according to the manufacturer's protocol.

The tissue sample was ground in liquid nitrogen until a fine powder was obtained using a mortar and pestle treated with DEPC. Approximately 100 milligrams of ground tissue was transferred to a sterile 1.5 ml Eppendorf tube. This sample tube was placed in liquid nitrogen until all samples were collected. Then, 450 µl of RLT buffer was added as provided in the kit (with the addition of mercaptoethanol) to each individual tube. The sample was vortexed vigorously and incubated at 56 ° C for 3 minutes. Then, the lysate was applied to the QIAshredder® spin column resting in a 2 ml collection tube, and centrifuged for 2 minutes at maximum speed. The unbound fraction was collected and ethanol volume was added 0.5 times to the clarified lysate. The mixture was mixed well and transferred to a Rneasy® mini-spin column that rests in a 2 ml collection tube. The sample was centrifuged for 1 minute at 10,000 rpm. Next, 700 µl of RW1 buffer was pipetted onto the Rneasy® column and centrifuged for 1 minute at

10,000 rpm RPE buffer was pipetted onto the Rneasy® column in a new collection tube and centrifuged

5 for 1 minute at 10,000 rpm. RPE buffer was added again to the Rneasy® spin column and centrifuged for 2 minutes at maximum speed to dry the membrane. To remove any remaining ethanol, the membrane was placed in a separate collection tube and centrifuged for an additional 1 minute at maximum speed. The Rneasy® column was transferred to a new 1.5 ml collection tube and 40 µl of RNase-free water was pipetted directly onto the Rneasy® membrane. This final elution tube was centrifuged for 1 minute at 10,000 rpm. The quality and quantity of total RNA was analyzed by gel with denatured formaldehyde and spectrophotometer.

Poly (A) RNA was isolated using the poly A + Oligotex® RNA purification kit (Qiagen Inc.) following the manufacturer's protocol. Approximately 200 µg of total RNA was used in 250 µl of maximum volume. A volume of 250 µl of OBB buffer was added and 15 µl of Oligotex® suspension was added to 250 µl of total RNA. The content was mixed thoroughly by pipetting and incubating for 3 minutes at 70 ° C on a heating block. The sample was then placed at room temperature for approximately 20 minutes. The Oligotex®: mRNA complex was pelleted by centrifugation for 2 minutes at maximum speed. Everything was removed except 50 µl of the supernatant from the microcentrifuge tube. The sample was further treated by OBB buffer. The Oligotex®: mRNA sediment was resuspended in 400 µl of OW2 buffer by vortexing. This mixture was transferred on a small centrifuge column placed in a new tube and centrifuged for 1 minute at maximum speed. The centrifuge column was transferred to a new tube and an additional 400 µl of OW2 buffer was added to the column. Then, the tube was centrifuged for 1 minute at maximum speed. The centrifuge column was transferred to a final 1.5 ml microcentrifuge tube. The sample was eluted with 60 µl of hot OEB buffer (70 ° C). The poly A product was analyzed by gels with

25 denatured formaldehyde and spectrophotometric analysis.

Example 3

PCR with reverse tanscription

First strand cDNA was produced using SuperScript reverse transcriptase following the manufacturer's protocol (Invitrogen, Carlsbad, California). The mixture of poly A + enriched RNA / oligo dT primer consisted of less than 5 µg of total RNA, 1 µl of 10 mM dNTP mixture, 1 µl of Oligo d (T) 12-18 (0.5 ! g /! l) and up to 10! l of water treated with DEPC. Each sample was incubated at 65 ° C for 5 minutes, then placed on ice for at least 1 minute. A reaction mixture was prepared by adding each of the following components in order: 2 µl of 10X RT buffer, 4 µl of 25 mM MgCl2, 2 µl of 0.1 M DTT and 1 µl of recombinant RNAse inhibitor RNase OUT. An addition of 9 µl of reaction mixture was pipetted to each RNA / primer mixture and mixed gently. Be

35 incubated at 42 ° C for 2 minutes and 1 µl of Super Script II RT was added to each tube. The tube was incubated for 50 minutes at 42 ° C. The reaction was terminated at 70 ° C for 15 minutes and cooled on ice. The sample was collected by centrifugation and 1 µl of RNase H was added to each tube and incubated for 20 minutes at 37 ° C. The second PCR was carried out with 200 pmoles of direct primer and 100 pmoles of reverse primer (mixture of oligo d (T) of 18 nt followed by 1 random base).

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

Example 4

45 Generation of PCR fragment populations

PCR fragments of Example 3 were ligated into a pGEM-T Easy vector (Promega, Madison, Wisconsin) following the manufacturer's instructions. The ligated product was transformed into competent JM109 cells and plated on plates with LB medium for blue / white selection. Colonies were selected and grown in a 96-well plate with 1.2 ml of LB medium overnight at 37 ° C. A frozen reserve was generated for all selected colonies. Plasmid DNA was purified from the plates using Beckman's Biomeck 2000 mini-preparation robotic systems with the Wizard SV Miniprep kit (Promega). Plasmid DNA was eluted with 100 µl of water and stored in a 96-well plate. Plasmids were digested by EcoR1 and analyzed using 1% agarose gel to confirm the amount of DNA and the size of the inserts. Plasmids containing a 400-600 bp insert were sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, California). The sequences were aligned with the GenBank database by BLAST search. Fragments related to p450 were identified and analyzed further. Alternatively, they were isolated

fragments of p450 from subtraction libraries. These fragments were also analyzed as described above.

Example 5

Construction of a cDNA library

A cDNA library was constructed by preparing total RNA from ethylene treated sheets as follows. First, total RNA was extracted from ethylene treated sheets of tobacco line 58-33 using an extraction protocol with chloroform and modified acid phenol. The protocol was modified to use a gram of tissue that was ground and subsequently vortexed in 5 ml of extraction buffer (100 mM Tris-HCl, pH 8.5; 200 mM NaCl; 10 mM EDTA; 0 SDS, 5%) to which 5 ml of phenol (pH 5.5) and 5 ml of chloroform were added. The extracted sample was centrifuged and the supernatant was stored. This extraction step was repeated 2-3 times until the supernatant appeared transparent. Approximately 5 ml of chloroform was added to remove trace amounts of phenol. RNA from the combined supernatant fractions was precipitated by adding a 3-fold volume of ethanol and 1/10 of the volume of 3M NaOAc (pH 5.2) and storing at -20 ° C for 1 hour. After transferring to a Corex glass vessel, the RNA fraction was centrifuged at 9,000 rpm for 45 minutes at 4 ° C. The sediment was washed with 70% ethanol and centrifuged for 5 minutes at 9,000 rpm at 4 ° C. After drying the sediment, the sedimented RNA was dissolved in 0.5 ml of RNase-free water. The quality and quantity of total RNA was analyzed by gel with denatured formaldehyde and spectrometry, respectively.

The resulting total RNA was used to isolate poly A + RNA using an oligo (dT) -cellulose (Invitrogen) protocol and microcentrifuge centrifuge columns (Invitrogen) by the following protocol. Approximately twenty mg of total RNA was subjected to purification twice to obtain high quality poly A + RNA. The poly A + RNA product was analyzed by gel analysis with denatured formaldehyde and subsequent RT-PCR of known full-length genes to ensure high mRNA quality.

Next, poly A + RNA was used as a template to produce a cDNA library using a cDNA synthesis kit, the ZAP-cDNA synthesis kit, and the ZAP-cDNA cloning kit Gigapack III gold (Stratagene, La Jolla, California). The method involved following the manufacturer's protocol as specified. Approximately 8 µg of poly A + RNA was used to construct a cDNA library. Analysis of the primary library revealed approximately 2.5 x 106 - 1 x 107 pfu. A background test of library quality was completed by complementation assays using IPTG and X-gal, in which recombinant plaques were expressed more than 100 times above the background reaction.

A more quantitative analysis of the library by random PCR showed that the average size of the insert cDNA was approximately 1.2 kb. The method used a two-stage PCR method. For the first stage, reverse primers were designed based on preliminary sequence information obtained from p450 fragments. Designed reverse primers and T3 (direct) primers were used to amplify corresponding genes from the cDNA library. The PCR reactions were subjected to agarose electrophoresis and cleaved, purified, cloned and the corresponding high molecular weight bands sequenced. In the second stage, new primers designed from the 5 'UTR or the initial coding region of p450 were used as direct primers together with the reverse primers (designed from the 3' UTR of p450) in the subsequent PCR obtaining full length p450 clones.

The p450 fragments were generated by PCR amplification from the constructed cDNA library as described in example 3 with the exception of the reverse primer. The T7 primer located on the plasmid in the 3 ′ sense of the cDNA inserts was used as the reverse primer. PCR fragments were isolated, cloned and sequenced as described in example 4.

Full length p450 genes were isolated by this PCR method from the constructed cDNA library. Inverse gene-specific primers (designed from the 3 ′ sense sequence of p450 fragments) and a direct primer (T3 in the library plasmid) were used to clone the full-length genes. PCR fragments were isolated, cloned and sequenced. If necessary, a second stage of PCR was applied. In the second stage, new direct primers designed from the 5 'UTR of the cloned P450 were used together with the inverse primers designed from the 3' UTR of p450 clones in subsequent PCR reactions to obtain clones. of full length p450. Subsequently the clones were sequenced.

Example 6

Cloned fragment characterization - reverse Southern type transfer analysis

Large-scale non-radioactive inverse Southern blot transfer assays were performed on all p450 clones identified in previous examples to detect differential expression. It was observed that the level of expression between different clusters of p450 was very different. Additional real-time detection was performed in those with high expression.

Southern non-radioactive transfer procedures were performed as follows.

1) Total RNA was extracted from converting (58-33) and non-converting sheets (58-25) treated and not treated with ethylene using the Riaeasy kit from Qiagen as described in example 2.

2) A probe was produced by biotin labeling a single stranded cDNA derived from poly A +-enriched RNA generated in the previous step. This single-stranded cDNA labeled by RT-PCR of the total converter and non-converting RNA (Invitrogen) was generated as described in example 3 with the exception of the use of biotinylated oligo dT as a primer (Promega). These were used as a probe to hybridize with the cloned DNA.

3) Plasmid DNA was digested with restriction enzyme EcoR1 and processed on agarose gels. The gels were dried simultaneously and transferred to two nylon membranes (Biodyne B). One membrane was hybridized with a converting probe and the other with a non-converting probe. The membranes were crosslinked with UV light (self-crosslinking parameter, 254 nm, Stratagene, Stratalinker) before hybridization.

Alternatively, the inserts were amplified by PCR from each plasmid using the sequences located in both arms of plasmid p-GEM, T3 and SP6, as primers. PCR products were analyzed by processing in 96-well Ready-to-run agarose gels. Confirmed inserts were subjected to timely transfer on two nylon membranes. One membrane was hybridized with a converting probe and the other with a non-converting probe.

4) The membranes were hybridized and washed according to the manufacturer's instructions with the modification of washing rigor (Enzo MaxSence kit, Enzo Diagnostics, Inc, Farmingdale, NY). The membranes were previously hybridized with hybridization buffer (2x SSC buffered formamide, containing detergent and hybridization enhancers) at 42 ° C for 30 min. and hybridized with 10 µl of denatured probe overnight at 42 ° C. Then, the membranes were washed with wash plug and 1X hybridization 1 time at room temperature for 10 min. and 4 times at 68 ° C for 15 min. The membranes were ready for the detection procedure.

5) The washed membranes were detected by alkaline phosphatase labeling by colorimetric detection with NBT / BCIP as described in the manufacturer's detection procedure (Enzo Diagnostics, Inc.). The membranes were blocked for one hour at room temperature with 1x blocking solution, washed 3 times with 1X detection reagents for 10 min., Washed twice with 1x pre-revealed reaction buffer for 5 min. and then the points in developing solution were revealed for 30-45 min. until the points appeared. The manufacturer (Enzo Diagnostics, Inc) provided all reagents. In addition, a large-scale inverse Southern type test was also performed using the KLP Southern detection and hybridization kit following the manufacturer's instructions (KPL, Gaithersburg, Maryland).

Example 7

Clone characterization - Northern type transfer analysis

As an alternative to Southern type transfer analysis, some membranes were hybridized and detected as described in the example of Northern type transfer assays. Northern type hybridization was used to detect differentially expressed mRNA in Nicotiana as follows.

A random priming method was used to prepare probes from cloned p450 (Megaprime DNA labeling systems, Amersham Biosciences). The following components were mixed: 25 ng denatured DNA template; 4 µl of each dTTP, dGTP and unmarked dCTP; 5 µl of reaction buffer; dATP marked with P32 and 2 µl of Klenow I; and H2O, to bring the reaction to 50 µl. The mixture was incubated at 37 ° C for 1-4 hours, and stopped with 2 µl of 0.5 M EDTA. The probe was denatured by incubation at 95 ° C for 5 minutes before use.

RNA samples were prepared from recent leaves treated and not treated with ethylene from several pairs of tobacco lines. In some cases, RNA enriched in poly A + was used. Approximately 15 µg of total RNA or 1.8 µg of mRNA (RNA and mRNA extraction methods as described in example 5) were brought to the same volume with DEPC H2O (5-10 µl). The same volume of loading buffer (1x MOPS; 18.5% formaldehyde; 50% formamide; 4% Ficoll 400; bromophenol blue) and 0.5 µL of EtBr (0.5 µg /) were added ! l). The samples were subsequently denatured in preparation for RNA separation by electrophoresis.

Samples were electrophoresed on a gel with formaldehyde (1% agarose, 1x MOPS, 0.6 M formaldehyde) with 1X MOP buffer (0.4 M morpholinopropanesulfonic acid; 0.1 M Na-acetate-3 x H2O; 10 mM EDTA; adjust to pH 7.2 with NaOH). RNA was transferred to a Hybond-N + membrane (nylon, Amersham Pharmacia Biotech) by the capillarity method in 10X SSC buffer (1.5 M NaCl; 0.15 M Na-citrate) for 24 hours. Samples of RNA membranes were crosslinked with UV (self-crosslinking parameter, 254 nm, Stratagene, Stratalinker) before hybridization.

The membrane was previously hybridized for 1-4 hours at 42 ° C with 5-10 ml of prehybridization buffer (5x SSC; 50% formamide; 5x Denhardt solution; 1% SDS; heat-denatured fragmented non-homologous DNA 1,00! g / ml) The old prehybridization buffer was discarded, and new prehybridization buffer and probe were added. Hybridization was carried out overnight at 42 ° C. The membrane was washed for 15 minutes with 2x SSC at room temperature, followed by a 2x SSC wash.

Northern type analysis was performed using full-length clones in tobacco tissue obtained from Burly converting and non-converting lines that were induced by ethylene treatment. The purpose was to identify the full-length clones that showed high expression in ethylene-induced converter lines with respect to ethylene-induced converter lines with respect to ethylene-induced Burley non-converting lines. By doing this, the functionality ratio of full length clones can be determined by comparing biochemical differences in leaf constituents between converting and non-converting lines.

Example 8

Immunodetection of p450 encoded by cloned genes

Peptide regions corresponding to 20-22 amino acids in length of three clones of p450 were selected so that 1) they had lower homology or did not have homology with other clones and 2) they had good hydrophilicity and antigenicity. The amino acid sequence of the selected peptide regions of the respective clones of p450 are listed below. Peptides synthesized with KHL were conjugated and then injected into rabbits. Antisera were collected 2 and 4 weeks after the 4th injection (Alpha Diagnostic Intl. 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 for cross-reactivity with target plant tissue proteins by Western blot analysis. Raw protein extracts were obtained from intermediate sheets treated with ethylene (from 0 to 40 hours) of converting and non-converting lines. Protein concentrations of the extracts were determined using RC DC protein assay kit (BIO-RAD) following the manufacturer's protocol.

Two micrograms of protein were loaded into each lane and the proteins were separated in gels with 10% -20% gradient using the Laemmli SDS-PAGE system. Proteins were transferred from the gels to PROTRAN nitrocellulose transfer membranes (Schleicher & Schuell) with the Trans-Blot Semi-Dry cell (BIO-RAD). Target p450 proteins were detected and visualized with the Western ECL Advance immunoblot detection kit (Amersham Biosciences). Primary antibodies against the synthetic peptide-KLH conjugates were prepared in rabbits. Secondary antibodies against rabbit IgG, coupled with peroxidase, were acquired from Sigma. Both primary and secondary antibodies were used at 1: 1000 dilutions. The antibodies showed strong reactivity against a single band in Western blots indicating that the antisera were monospecific with respect to the target peptide of interest. The antisera also cross-reacted with synthetic peptides conjugated to KLH.

Example 9

Nucleic acid identity and structure relationship of isolated nucleic acid fragments

More than 100 cloned p450 fragments were sequenced in conjunction with the Northern-type transfer analysis to determine their structural relationship. The approach used direct primers based on any of two common p450 motifs located near the carboxyl terminus of the p450 genes. Direct primers corresponded to cytochrome p450 FXPERF (SEQ ID NO: 11) or GRRXCP (A / G) (SEQ ID NO: 12) motifs. The reverse primers used conventional primers of either the plasmid, SP6 or T7 located on both arms of the pGEM plasmid, or a poly A tail. The protocol used is described below.

Spectrometry was used to estimate the concentration of the starting double stranded DNA following the manufacturer's protocol (Beckman Coulter). The mold was diluted with water to the appropriate concentration, denatured by heating at 95 ° C for 2 minutes and then placed on ice. The sequencing reaction was prepared on ice using 0.5 to 10 µl of denatured DNA template, 2 µl of 1.6 pmoles of the direct primer, 8 µl of DTCS Quick Start Master Mix and the total volume was carried up to 20 µl with water. The thermocycling program consisted of 30 cycles of the following cycle: 96 ° C for 20 seconds, 50 ° C for 20 seconds and 60 ° C for 4 minutes followed by maintenance at 4 ° C.

The sequencing reaction was stopped by adding 5 µl of stop buffer (equal volume of 3M NaOAc and 100 mM EDTA and 1 µl of 20 mg / ml glycogen). The sample was precipitated with 60 µl of cold 95% ethanol and centrifuged at 6000 x g for 6 minutes. The ethanol was discarded. The sediment was washed twice with 200 µl of 70% cold ethanol. After drying the sediment, 40 µl of SLS solution was added and the sediment was resuspended. It was covered with a layer of mineral oil. Then, the sample was placed in the CEQ 8000 automated sequencer for further analysis.

In order to verify the nucleic acid sequences, the nucleic acid sequence was re-sequenced in both directions using direct primers for the FXPERF region (SEQ ID NO: 11) or GRRXCP (A / G) (SEQ ID NO: 12 ) of the p450 gene or reverse primers for either the plasmid or the poly A tail. All sequencing was carried out at least twice in both directions.

The nucleic acid sequences of cytochrome p450 fragments were compared with each other from the coding region corresponding to the first nucleic acid after the region coding for the GRRXCP (A / G) motif (SEQ ID NO: 12) to the termination codon. This region was selected as an indicator of genetic diversity among p450 proteins. A large number of genetically distinct p450 genes were observed, in excess of 70 genes, similar to other plant species. With the comparison of nucleic acid sequences, it was found that genes could be placed in different groups of sequences based on their sequence identity. It was found that the best single cluster of p450 members was determined to be sequences with a nucleic acid identity of 75% or higher. (See, for example, table 1 of patent application publication US 2004/0162420).

The reduction in the percentage of identity resulted in significantly larger groups. A preferred cluster was observed for sequences with a nucleic acid identity of 81% or higher, a more preferred cluster with a nucleic acid identity of 91% or higher and a most preferred cluster for sequences with a nucleic acid identity 99% or higher. The majority of the groups contained at least two members and often three or more members. Others were not discovered repeatedly, suggesting that the approach taken could isolate mRNA from both high and low expression in the tissue used.

Using gene chip technology to identify genes that are differentially expressed in converting versus non-converting tobacco lines, it was determined that D121-AA8 had reproducible induction in converter lines treated with ethylene. Based on these results, the D121-AA8 gene (whose cDNA sequence is the sequence of SEQ ID NO: 3; Figure 3) was identified as the tobacco nicotine demethylase gene of interest.

In view of the p450 nomenclature rule, the tobacco nicotine demethylase gene is novel and belongs to the CYP82E class (The Arabidopsis Genome Initiative (AGI) and The Arabidopsis Information Resource (TAIR); Frank, Plant Physiol. 110: 1035 -1046, 1996; Whitbred et al., Plant Physiol. 124: 47-58, 2000); Schopfer 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 analyzes, for example, as described in previously submitted applications, determined that the sequence of SEQ ID NO: 3 encodes a tobacco nicotine demethylase (SEQ ID NO: 4; Figure 3).

In particular, the function of the candidate clone (D121-AA8), as the coding gene for nicotine demethylase, was confirmed by testing the enzymatic activity of p450 expressed heterologously in yeast cells as follows.

1. Construction of the yeast expression vector

The supposed protein coding sequence of the cDNA encoding tobacco nicotine demethylase (D121AA8) was cloned into the yeast expression vector pYeDP60. Appropriate BamHI and MfeI sites (underlined below) were introduced by PCR primers containing these sequences either in the 5 'direction of the translation start codon (ATG) or in the 3' direction of the termination codon (TAA). The MfeI in the amplified PCR product is compatible with the EcoRI site in the vector. The primers used to amplify the cDNA were 5'-TAGCTACGCGGATCCATGCTTTCTCCCATAGAAGCC-3 '(SEQ ID NO: 27) and 5'-CTGGATCACAATTGTTAGTGATGGTGATGGTGATGCGATCCTCTATAAAG CTCAGGTGCCAGGC-3' (SEQ ID NO: 28). A sequence segment coding for nine extra amino acids was incorporated into the C-terminal end of the protein, which includes six histidines, in the reverse primer to facilitate the expression of p450 labeled with 6-His after induction. The PCR products in the YeDP60 vector were ligated after enzymatic digestions in the sense orientation with reference to the GAL10-CYC1 promoter. Appropriate construction of the yeast expression vector was verified by restriction enzyme analysis and DNA sequencing.

2.

Yeast Transformation

The WAT11 yeast line, modified to express NADPH-cytochrome p450 reductase ATR1 from Arabidopsis, was transformed with the cDNA plasmids pYeDP60-p450. Fifty microliters of WAT11 yeast cell suspension was mixed with !1 µg of plasmid DNA in a 0.2 cm electrode separating cell. A pulse at 2.0 kV was applied using an Eppendorf electroporator (model 2510). Cells were spread on SGI plates (bactocasamino acids 5 g / l, nitrogenous yeast base without amino acids 6.7 g / l, glucose 20 g / l, DL-tryptophan 40 mg / l, agar 20 g / l). Transformants were confirmed by PCR analysis carried out directly in randomly selected colonies.

3.

 Expression of p450 in transformed yeast cells

Individual yeast colonies were used to inoculate 30 ml of SGI medium (bactocasamino acids 5 g / l, nitrogenous yeast base without amino acids 6.7 g / l, glucose 20 g / l, DL-tryptophan 40 mg / l) and made grow at 30 ° C for approximately 24 hours. An aliquot of this culture was diluted 1:50 in 1000 ml of YPGE medium (yeast extract 10 g / l, bactopeptone 20 g / l, glucose 5 g / l, ethanol 30 ml / l) and grown until it was completely consumed glucose as indicated by the colorimetric change of a Diastix urinalysis test strip (Bayer, Elkhart, IN). The induction of cloned p450 was started by adding DL-galactose to a final concentration of 2%. Cultures were grown for an additional 20 hours before being used for in vivo activity testing or for the preparation of microsomes.

They used WAT11 yeast cells that express pYeDP60-CYP71D20 (a p450 that catalyzes the hydroxylation of 5-epi-aristoloquene and 1-deoxycoxysidiol in Nicotiana tabacum) as a control for p450 expression and enzyme activity assays.

To evaluate the efficacy of yeast expression of p450 in great detail, reduced CO difference spectroscopy was performed. The reduced CO spectrum showed a peak at 450 nm of proteins from the four yeast lines transformed with p450. No similar peaks were observed in the microsomes of the control yeast or vector control yeast. The results indicated that p450 proteins were effectively expressed in the yeast lines that harbored the pYeDP60-CYP 450. The concentrations of p450 protein expressed in yeast microsome ranged from 45 to 68 nmoles / mg of total protein.

4. Enzyme assay in vivo

Nicotine demethylase activity in the transformed yeast cells was tested by feeding the yeast culture with DL-Nicotine (pyrrolidine-2-14C). 14C-labeled nicotine (54 mCi / mmol) was added to 75 µl of the galactose-induced culture to provide a final concentration of 55 µM. The test culture was incubated with shaking in 14 ml polypropylene tubes for 6 hours and extracted with 900 µl of methanol. After centrifuging, 20 µl of the methanol extract was separated with a rp-HPLC and the nornicotine fraction was quantified by LSC.

The control culture of WAT11 (pYeDP60-CYP71D20) did not convert nicotine into nornicotine, which shows that the yeast strain WAT11 does not contain endogenous enzymatic activities that can catalyze the bioconversion stage of nicotine into nornicotine. In contrast, the yeast expressing the tobacco nicotine demethylase gene produced a detectable amount of nornicotine, indicating the nicotine demethylase activity of the translation product of SEQ ID NO: 3.

5. Preparation of yeast microsomes

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

5 microsomals in a liquid nitrogen freezer until use.

6. Enzyme activity test in microsomal yeast preparations

Nicotine demethylase activity assays were carried out with microsomal yeast preparations. In particular, DL-Nicotine (pyrrolidine-2-14C) was obtained from Moravek Biochemicals and had a specific activity of 54 mCi / mmol. Chlorpromazine (CPZ) and oxidized cytochrome c (cit. C), both inhibitors of P450, from Sigma were purchased. 10 The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) is the typical electron donor for cytochrome P450 through NADPH: cytochrome P450 reductase. NADPH was omitted for control incubation. The routine enzyme assay included microsomal proteins (approximately 1 mg / ml), 6 mM NADPH and nicotine labeled with 14C 55 µM. The concentration of CPZ and cit. C, when used, was 1 mM and 100 µM, respectively. The reaction was carried out at 25 ° C for 1 hour and stopped with the addition of 300 µl of methanol 15 to each 25 µl reaction mixture. After centrifugation, 20 µl of the methanol extract was separated with a reverse phase high performance liquid chromatography (HPLC) (Agilent) system using an Inertsil ODS-3 3! (150 x 4.6 mm) from Varian. The isocratic mobile phase was the mixture of methanol and 50 mM potassium phosphate buffer, pH 6.25, with a ratio of 60:40 (v / v) and the flow rate was 1 ml / min. The nornicotine peak was collected, as determined by comparison with authentic unlabeled nornicotine and

20 was subjected to 2900 tri-carb liquid scintillation counter (LSC) (Perkin Elmer) for quantification. The activity of nicotine demethylase was calculated based on the production of nornicotine labeled with 14C for 1 hour of incubation.

Microsomal preparations from control yeast cells expressing CYP71D20 had no microsomal nicotine demethylase activity. In contrast, microsomal samples obtained from yeast cells that expressed the tobacco nicotine demethylase gene showed significant levels of nicotine demethylase activity. Nicotine demethylase activity required NADPH and was shown to be inhibited by specific inhibitors of p450, which is consistent with the fact that tobacco nicotine demethylase is a p450. The enzymatic activity for tobacco nicotine demethylase (D121-AA8) was approximately 10.8 pKat / mg protein as calculated by radioactive intensity and protein concentrations. A typical set of

30 results of enzymatic assays obtained for yeast cells are shown in table 1.

TABLE 1: DEMTILLATION ACTIVITY IN MICROSOMAS OF YEAST CELLS EXPRESSING D121-AA8 and P450 CONTROL

Sample

Microsomes Microsomes + 1 mM chlorpromazine Microsomes + cytochrome C 100! M -NADPH microsomes

D121-AA8

10.8 ± 1.2 * pkat / mg protein 1.4 ± 1.3 pkat / mg protein 2.4 ± 0.7 pkat / mg protein 0.4 ± 0.1 pkat / mg protein

Control (CYP71D20)

Not detected Not detected Not detected Not detected

* Average results of 3 replicas.

Together, these experiments demonstrated that full-length tobacco nicotine demethylase

Cloned (SEQ ID NO: 3; D121-AA8) encodes a cytochrome p450 protein that catalyzes the conversion of nicotine to nornicotine when expressed in yeast.

Example 11

Related amino acid sequence identity of isolated nucleic acid fragments

The amino acid sequences of nucleic acid sequences obtained for fragments of

Cytochrome p450 of Example 8. The deduced region corresponded to the amino acid immediately after the sequence motif GXRXCP (A / G) (SEQ ID NO: 13) to the carboxyl terminus, or termination codon. When comparing the sequence identity of the fragments, a unique grouping was observed for the sequences with an amino acid identity of 70% or higher. A preferred grouping was observed for sequences with an amino acid identity of 80% or higher, more preferred with an amino acid identity of 90% or higher, and a most preferred grouping for sequences with an amino acid identity of 99% or higher. It was found that several of the unique nucleic acid sequences have complete amino acid identity with respect to other fragments and therefore only one member with the identical amino acid was reported.

5 At least one member of each amino acid identity group was selected for functional and gene cloning studies using plants. In addition, members of the groups that were differentially affected by treatment with ethylene or other biological differences were selected as assessed by Northern and Southern analysis for functional and gene cloning studies. To aid in gene cloning, expression studies and evaluations of whole plants, peptide specific antibodies can be prepared based on sequence identity and differential sequence.

Example 12

Related amino acid sequence identity of full length clones

The nucleic acid sequence of full length Nicotiana genes cloned in Example 5 was deduced to determine its entire amino acid sequence. Cytochrome p450 genes were identified by the presence of three three conserved p450 domain motifs, corresponding to UXXRXXZ (SEQ ID NO: 14), PXRFXF (SEQ ID NO: 15) or GXRXC (SEQ ID NO: 16) at the end carboxyl terminal in which U is E or K, X is any amino acid and Z is P, T, S or M. All p450 genes were characterized to determine their amino acid identity using a BLAST program comparing their full length sequences each other and against known tobacco genes. The program used the NCBI special BLAST tool (two sequence alignment (b12seq), http://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html). Two sequences were aligned in BLASTN without filter for nucleic acid sequences and BLASTP for amino acid sequences. Based on their percentage of amino acid identity, each sequence was grouped into identity groups in which the grouping contained members who shared an identity of at least 85% with another member. A preferred cluster was observed for sequences with an amino acid identity of 90% or higher, a more preferred cluster

25 had an amino acid identity of 95% or higher and a most preferred grouping had sequences with an amino acid identity of 99% or higher. It was deduced that the amino acid sequence of the full-length nicotine demethylase gene had the sequence provided in SEQ ID NO: 4 (Figure 3).

Example 13

Nicotiana cytochrome p450 clones lacking one or more of the specific P450 domains of tobacco

Four clones had high nucleic acid homology, which ranged between 90% and 99% nucleic acid homology. However, due to a shift in the nucleotide reading frame these genes did not contain one or more of the three cytochrome p450 domains of the C-terminus and were excluded from the identity groups.

Example 14

35 Use of Nicotiana cytochrome p450 fragments and clones in the altered regulation of tobacco qualities

The use of p450 nucleic acid fragments or whole tobacco genes is useful in the identification and selection of plants that have altered tobacco phenotypes or constituents of tobacco and, more importantly, altered metabolites. Transgenic tobacco plants are generated by a variety of transformation systems that incorporate nucleic acid fragments or full length genes, selected from those reported herein, in orientations for either regulation by decrease, for example antisense orientation, or overexpression, for example, sense orientation and the like. For overexpression in full length genes, any nucleic acid sequence encoding all or a functional part or amino acid sequence of the full length genes described in this invention is desirable. Such nucleic acid sequences are desirably effective in increasing the expression of a given

Enzyme and therefore result in a phenotypic effect within Nicotiana. Nicotiana lines that are homozygous are obtained through a series of backcrossing and are evaluated to determine phenotypic changes including, but not limited to, endogenous p450 RNA analysis, transcripts, expressed p450 peptides and plant metabolite concentrations using commonly used techniques. available to a person skilled in the art. The changes presented in tobacco plants provide information on the functional role of the gene of interest selected or are of use as preferred Nicotiana plant species.

Example 15

Cloning of genomic tobacco nicotine demethylase from Burley tobacco converter

Genomic DNA from Burley converting tobacco plant line 4407-33 (a 4407 line of Nicotiana tabacum variety) was extracted using the Qiagen Plant Easy kit as described in the previous examples (see also the manufacturer's procedure).

5 Primers were designed based on promoter region 5 ’and UTR in 3’ cloned in previous examples. The direct primers were 5'-GGC TCT AGA TAA ATC TCT TAA GTT ACT AGG TTC TAA-3 '(SEQ ID NO: 17) and 5'-TCT CTA AAG TCC CCT TCC-3' (SEQ ID NO: 25) and the reverse primers were 5'-GGC TCT AGA AGT CAA TTA TCT TCT ACA AAC CTT TAT ATA TTA GC-3 '(SEQ ID NO: 18), and 5'-CCA GCA TTC CTC AAT TTC-3' (SEQ ID NO : 26). PCR was applied to genomic DNA 4407-33 with 100 µl of reaction mixture. The high fidelity enzyme Pfx was used for PCR amplification. The 1% agarose gel PCR product was visualized after electrophoresis. A single band with a molecular weight of approximately 3.5 kb was observed and cut from the gel. The resulting band was purified using a gel purification kit (Qiagen; based on the manufacturer's procedure). The purified DNA was digested by the enzyme Xba I (NEB; used according to the manufacturer's instructions). Plasmid pBluescript was digested with Xba I using the same procedure. The gel was purified on

15 fragment and ligated into plasmid pBluescript. The ligation mixture was transformed into the competent GM1O9 cell and plated onto LB plates containing 100 mg / l ampicillin with blue / white selection. The white colonies were collected and grown in 10 ml of LB liquid medium containing ampicillin. The 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 procedure. Be the T3 and T7 primers and 8 other internal primers to sequence. The sequence was assembled and analyzed, thus providing the genomic sequence (SEQ ID NO: 1; Figures 2-1 to 2-3).

The comparison of the sequence of SEQ ID NO: 1 with the sequence of SEQ ID NO: 3 allowed the determination of a single intron within the coding part of the gene (identified as the sequence of SEQ ID NO: 5; Figure 4). As shown in Figure 1, the genomic structure of tobacco nicotine demethylase includes two exons that

25 flank a single intron. The first exon encompasses nucleotides 2010 to 2949 of SEQ ID NO: 1, which code for amino acids 1-313 of SEQ ID NO: 2, and the second exon encompasses nucleotides 3947 to 4562 of SEQ ID NO: 1, which encode for amino acids 314-517 of SEQ ID NO: 2. Accordingly, the intron encompasses nucleotides 2950-3946 of SEQ ID NO: 1. The intron sequence is provided in Figure 4 and is that of SEQ ID NO: 5. The translation product of the genomic DNA sequence is provided in Figure 2-1 as the sequence of SEQ ID NO: 2. The amino acid sequence of tobacco nicotine demethylase contains a reason for anchoring to the endoplasmic reticulum membrane.

Example 16

Cloning of flanking sequences in 5 ’(SEQ ID NO: 6) and UTR in 3’ (SEQ ID NO: 7) from tobacco converter

35 A. Isolation of total DNA from tissue of converting tobacco leaves

Genomic DNA was isolated from tobacco leaves converter 4407-33. DNA isolation was carried out using a DNeasy Plant Mini kit from Qiagen, Inc. (Valencia, Ca) according to the manufacturer's protocol (see manufacturer's manual Dneasy 'Plant Mini and DNeasy Plant Maxi Handbook, Qiagen January 2004 ). The procedure for DNA preparation included the following steps: tobacco leaf tissue (approximately 20 mg dry weight) was ground to give a fine powder under liquid nitrogen for 1 minute. The tissue powder was transferred to a 1.5 ml tube. AP1 buffer (400 µl) and 4 µl of RNase stock solution (100 mg / ml) were added to a maximum of 100 mg of ground leaf tissue and vortexed vigorously. The mixture was incubated for 10 min. at 65 ° C and mixed 2-3 times during incubation by inverting the tube. Then, AP2 buffer (130 µl) was added to the lysate. The mixture was mixed and incubated for 5 min. on ice. Lysate 45 was applied to a QIAshredder Mini Spin column and centrifuged for 2 min. (14,000 rpm). The non-retained fraction was transferred to a new tube without altering the sediment of cell debris. Then, AP3 / E buffer (1.5 volumes) was added to the clarified lysate and mixed with pipetting. The mixture (650 µl) of the preceding step was applied including any precipitate to a DNeasy Mini Spin column. The mixture was centrifuged for 1 min. at> 6000 x g (> 8000 rpm) and the non-retained fraction was discarded. This was repeated with the remaining sample and the non-retained fraction and the collection tube were discarded. The DNeasy Mini Spin column was placed in a new 2 ml collection tube. Then, AW buffer (500 µl) was added to the DNeasy column and centrifuged for 1 min. (> 8000 rpm). The non-retained fraction was discarded. The collection tube was reused in the next stage. Then, AW buffer (500 µl) was added to the DNeasy column and centrifuged for 2 min. (> 14,000 rpm) in order to dry the membrane. The DNeasy column was transferred to a 1.5 ml tube. Then, AE buffer (100 µl) was pipetted onto the DNeasy membrane. The mixture was incubated for 5 min. at room temperature (15-25 ° C) and then centrifuged

for 1 min. (> 8000 rpm) to elute.

The quality and quantity of DNA was estimated by processing the samples on an agarose gel.

B. Cloning of 5 ’flanking sequences of the structural gene

A modified reverse PCR method was used to clone 750 nucleotides of the 5 ’flanking sequences of the structural gene of SEQ ID NO: 1. First, appropriate restriction enzymes were selected based on the restriction site in the fragment of known sequence and the distance of the restriction sites in the 3 'sense of the 5' flanking sequences. Primers were designed based on this known fragment. The direct primer was located in the 3 ’direction of the reverse primer. The reverse primer was placed in the 3 ’part of the known fragment.

The cloning procedure included the following stages:

The purified genomic DNA (5 µg) was digested with 20-40 units of the appropriate restriction enzyme (EcoRI and SpeI) in a 50 µl reaction mixture. An agarose gel electrophoresis was carried out with a 1/10 volume of the reaction mixture to determine if the DNA was digested to the end. Direct ligation was carried out after complete digestion by overnight ligation at 4 ° C. A reaction mixture of 200 µl containing 10 µl of digested DNA and 0.2 µl of T4 DNA ligase (NEB) was ligated overnight at 4 ° C. PCR was carried out on the ligation reaction after obtaining a small artificial circular genome. PCR was carried out with 10 µl of ligation reaction and 2 primers of known fragments in two different directions in 50 µl of reaction mixture. A gradient PCR program with mating temperatures of 45-56 ° C was applied.

Agarose gel electrophoresis was performed to check the PCR reaction. The desired gel band was cut and a QIAquick gel purification kit from QIAGEN was used to purify the band. Purified PCR fragments were ligated into a pGEM-T Easy vector (Promega, Madison, WI) following the manufacturer's instructions. Transformed DNA plasmids were extracted by miniprep. using the SV Miniprep kit (Promega, Madison, WI) following the manufacturer's instructions. Plasmid DNA containing the insert was sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, CA). Approximately 758 nt (nucleotides 1241-2009 of SEQ ID NO: 1) of the 5 ′ flanking sequence were cloned by the method described above.

C. Cloning of 5 ’longer flanking sequences (SEQ ID NO: 6; figure 5) of the structural gene

The BD GenomeWalker Universal kit (Clontech laboratories, Inc., PaloAlto, CA) was used to clone an additional 5 'flanking sequence of the structural gene, D121-AA8 according to the manufacturer's user manual (see manufacturer's manual BD GenomeWalker August of 2004). The size and purity of the genomic tobacco DNA were tested by processing samples on a 0.5% agarose gel. A total of 4 blunt end reactions (DRA I, STU I, ECOR V, PVU II) were established for the construction of a genomic walkway of 33 tobacco libraries. After purification of the digested DNAs, the digested genomic DNAs were ligated to the genomic path adapter. Primary PCR reactions were applied to the four digested DNAs using AP1 adapter primer and the D121-AA8 gene specific primer (CTCTATTGATACTAGCTGGTTTTGGAC; SEQ ID NO: 19). Primary PCR products were used directly as templates for nested PCR. The nested adapter primer provided by the kit and the nested primer of known clone D121-AA8 (SEQ ID NO: 3) (GGAGGGAGAGTATAACTTACGGATTC; SEQ ID NO: 20) were used in the PCR reaction. PCR products were checked by gel electrophoresis processing. The desired bands were cut from the gel, and the PCR fragments were purified using the QIAquick gel purification kit from QIAGEN. The purified PCR fragments were ligated into a pGEM-T Easy vector (Promega, Madison, WI) following the manufacturer's instructions. Transformed DNA plasmids were extracted by miniprep. using the SV Miniprep kit (Promega, Madison, WI) and following the manufacturer's instructions. Plasmid DNA containing the insert was sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, CA). Approximately 853 nt of the 5 ’flanking sequence was cloned, including nucleotides 399-1240 of SEQ ID NO: 1, by the method described above.

A second round of the genomic walk was performed according to the same method with the difference that the following primers GWR1A (5'-AGTAACCGATTGCTCACGTTATCCTC-3 ') (SEQ ID NO: 21) and GWR2A (5'-CTCTATTCAACCCCACACGGACTACT-3') were used (SEQ ID NO: 22). Approximately 398 nt of the flanking sequence, including nucleotides 1-398 of SEQ ID NO: 1, were cloned by this method.

A search for regulatory elements revealed that, in addition to the “TATA” box, the “CAAT” boxes and the “GAGA” boxes, several recognition sites similar to MYB and organ specificity elements are present in the nicotine promoter region tobacco demethylase Also present are stimulatory response elements and nitrogen-regulated elements, identified using conventional methods, in the promoter region.

D. Cloning of 3 ’flanking sequences of the structural gene

The BD GenomeWalker 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 user manual. The cloning procedure is the same as described in the preceding section C of this example, except for gene specific primers. The first primer was designed from the proximities to the end of the structural gene D121-AA8 (5’-CTA AAC TCT GGT CTG ATC CTG ATA CTT-3 ’) (SEQ ID NO: 23). The nested primer was further designed in the 3 ’sense of primer 1 of structural gene D 121-AA8 (CTA TAC GTA AGG TAA ATC CTG TGG AAC) (SEQ ID NO: 24). Final PCR products were checked by gel electrophoresis. The desired bands of the gel were cut. The PCR fragments were purified using the QIAquEN 10 QIAquick gel purification kit. The purified PCR fragments were ligated into a pGEM-T Easy vector (Promega, Madison, WI) following the manufacturer's instructions. Transformed DNA plasmids were extracted by miniprep. using the SV Miniprep kit (Promega, Madison, WI) following the manufacturer's instructions. Plasmid DNA containing the insert was sequenced using a CEQ 2000 sequencer (Beckman, Fullerton, CA). Approximately 1617 nucleotides of the flanking sequence were cloned into an additional 3 ’(nucleotides 4731-6347

SEQ ID NO 15: 1) by the method described above. The nucleic acid sequence of the UTR region at 3 ’is shown in Figure 6.

WO 03/078577, WO 2004/035745, PCT / US / 2004/034218 and PCT / US / 2004/034065 are cited herein.

Sequence list

20 <110> U. S. Smokeless Tobacco Company et al.

<120> Genomic clone of tobacco nicotine demethylase and uses thereof

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<150> Document PCT / US04 / 034218

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<160> 29 5 <170> PatentIn version 3.3

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<213> Tobacco Nicotine Demethylase Gene Promoter

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<213> UTR in 3 ’of the tobacco nicotine demethylase gene

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<223> Synthesized Peptide

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5 <210> 9

<211> 20

<212> PRT

<213> Artificial sequence

<220> 10 <223> Synthesized Peptide

<400> 9

<210> 10

<211> 22 15 <212> PRT

<213> Artificial sequence

<220>

<223> Synthesized Peptide

<400> 10

<210> 11

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Consensus sequence of the preserved p450 motif

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<221> MISC_FEATURE

<222> (2) .. (2)

<223> Xaa = any amino acid

<400> 11

<210> 12

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Consensus sequence of the preserved p450 motif

<220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> Xaa = any amino acid

<220>

<221> MISC_FEATURE

<222> (7) .. (7)

<223> Xaa = Ala or Gly

<400> 12

<210> 13

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<212> PRT

<213> Artificial sequence

<220>

<223> Consensus sequence of the preserved p450 motif

<220>

<221> MISC_FEATURE

<222> (2) .. (2)

<223> Xaa = any amino acid

<220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> Xaa = any amino acid

<220>

<221> MISC_FEATURE

<222> (7) .. (7)

<223> Xaa = Ala or Gly

<400> 13

<210> 14

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Consensus sequence of the preserved p450 motif

<220>

<221> MISC_FEATURE

<222> (1) .. (1)

<223> Xaa = Glu or Lys

<220>

<221> MISC_FEATURE

<222> (2) .. (3)

<223> Xaa = any amino acid

<220>

<221> MISC_FEATURE

<222> (5) .. (6)

<223> Xaa = any amino acid

<220>

<221> MISC_FEATURE

<222> (7) .. (7)

<223> Xaa = Pro, Thr, Ser, or Met

<400> 14

<210> 15

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Consensus sequence of the preserved p450 motif

<220>

<221> MISC_FEATURE

<222> (2) .. (2)

<223> Xaa = any amino acid

<220>

<221> MISC_FEATURE

<222> (5) .. (5)

<223> Xaa = any amino acid

<400> 15 <210> 16

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Consensus sequence of the preserved p450 motif

<220>

<221> MISC_FEATURE

<222> (2) .. (2)

<223> Xaa = any amino acid

<220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> Xaa = any amino acid

<400> 16

<210> 17

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 17

<210> 18

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 18

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<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 19

<210> 20

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 20

<210> 21

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 21

<210> 22

<211> 25 <212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 22

<210> 23

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 23

<210> 24

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 24

<210> 25

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 25

<210> 26

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 26

<210> 27

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 27

<210> 28

<211> 64

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 28

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence

<220> <223> Consensus sequence for reason p450 preserved

<220>

<221> MISC_FEATURE

<222> (2) .. (2) 5 <223> Xaa = any amino acid

<220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> Xaa = any amino acid 10 <220>

<221> MISC_FEATURE

<222> (6) .. (6)

<223> Xaa = any amino acid

<220> 15 <221> MISC_FEATURE

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<400> 29

Claims (14)

1. Isolated nucleic acid molecule encoding a nicotine demethylase, said nucleic acid molecule comprising (a) the sequence of SEQ ID NO: 1, or (b) at least 200 consecutive base pairs identical to 200 consecutive base pairs SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. The isolated nucleic acid molecule according to claim 1,

(to)

 said nucleic acid molecule being DNA;

(b)

 said nucleic acid molecule comprising the sequence of SEQ ID NO: 1;

(C)

 encoding said nucleic acid molecule for a nicotine demethylase comprising the amino acid sequence of SEQ ID NO: 2;

10 (d) said isolated nucleic acid molecule being operatively linked to a functional promoter in a plant cell; or (e) comprising a promoter that hybridizes under stringent conditions with the sequence of SEQ ID NO: 6, or a fragment comprising at least 200 consecutive base pairs identical to 200 consecutive base pairs of SEQ ID NO: 6, in which said promoter directs transcription, particularly in which the promoter is induced 15 after treatment with ethylene or during senescence. 3. Isolated nucleic acid according to claim 1 or 2,

(to)

 in which the expression of nicotine demethylase is induced after treatment with ethylene or during senescence; or

(b)

 wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 6.

The isolated nucleic acid molecule according to claim 1, the nucleic acid molecule comprising the sequence of SEQ ID NO: 5 or a fragment having at least 200 consecutive base pairs that can be obtained from SEQ ID NO: 5 . 5. The isolated nucleic acid molecule according to claim 1, the nucleic acid molecule comprising the sequence of SEQ ID NO: 7 or a fragment having at least 200 consecutive base pairs that can be obtained from SEQ ID NO: 7. 6. A transgenic tobacco plant having a reduced expression or altered enzymatic activity with respect to the level in a control plant of a nicotine demethylase encoded by a nucleic acid molecule according to claim 1, (a) said transgenic plant comprising a transgene having a sequence identity of 91% or higher 30 with SEQ ID NO: 1, or with a fragment thereof, which, when expressed in said transgenic plant, silences the gene expression of an endogenous tobacco nicotine demethylase; (b) said transgenic plant comprising a transgene that expresses an antisense molecule of a tobacco nicotine demethylase; (c) said transgenic plant comprising a transgene encoding a double stranded RNA molecule of a tobacco nicotine demethylase;

(d)

 said transgenic plant comprising a transgene which, when expressed in said transgenic plant, jointly suppresses the expression of a tobacco nicotine demethylase; or

(and)

 said transgenic plant comprising a deletion in an endogenous nicotine demethylase encoded by the sequence of SEQ ID NO: 1; Y

40 wherein said reduced expression or altered enzyme activity reduces the level of nornicotine in said plant.

7.

A transgenic tobacco plant according to claim 6, wherein said nicotine demethylase comprises the amino acid sequence of SEQ ID NO: 2.

8.

Vegetable component of the tobacco plant according to claim 6, particularly said plant component being a seed, stem or leaf of tobacco, especially in which said leaf is cured.

9.

Tobacco product comprising the vegetable component according to claim 8.

10. The tobacco plant seed according to claim 6, said seed comprising said transgene or said deletion. 11. Method of producing a tobacco nicotine demethylase, said method comprising the steps of:

(to)

 providing a cell transformed with an isolated nucleic acid molecule according to claim 1;

(b)

 culturing said transformed cell under conditions to express said isolated nucleic acid molecule; Y

(c) recover said tobacco nicotine demethylase. 12. Recombinant tobacco nicotine demethylase produced according to the method of claim 11. 13. Method of isolating a tobacco nicotine demethylase or fragment having at least 200 consecutive base pairs thereof, said method comprising the steps of: (a) contacting the nucleic acid molecule according to claim 1 with a nucleic acid preparation of a plant cell under hybridization conditions that provide for the detection of nucleic acid sequences 15 having a sequence identity of at least 70% or greater with a nucleic acid according to claim 1; Y (b) isolate said hybridizing nucleic acid sequences. 14. Method of isolation of a nucleic acid comprising SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, or fragment having at least 200 consecutive base pairs thereof, said method comprising the steps 20 of:

(to)

 provide a nucleic acid preparation from a plant cell;

(b)

 contacting a pair of oligonucleotides having sequence identity with a region of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 with said nucleic acid preparation under conditions suitable for reaction-mediated DNA amplification polymerase chain to produce a reaction product in

25 polymerase chain; Y (c) isolate the nucleic acid or fragment thereof produced in step (c).

fifteen.

Method according to claim 14, wherein said nucleic acid preparation is genomic DNA.

16.

Method according to claim 14, wherein said nucleic acid preparation is cDNA.

17.

Isolated nucleic acid molecule comprising SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

18. An isolated nucleic acid molecule according to claim 17, said isolated nucleic molecule being operably linked to a heterologous nucleic acid sequence.