Alteration of tobacco alkaloid content through modification of specific cytochrome P450 genes

Patent metadata

Field	Value
Publication number	US-9913451-B2
Application number	US-201514878083-A
Country	US
Kind code	B2
Filing date	Oct 8, 2015
Priority date	Feb 23, 2005
Publication date	Mar 13, 2018
Grant date	Mar 13, 2018

Abstract

Compositions and methods for reducing the level of nornicotine and N′-nitrosonornicotine (NNN) in Nicotiana plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for cytochrome P450s that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Expression cassettes, vectors, plants, and plant parts thereof comprising inhibitory sequences that target expression or function of the disclosed cytochrome P450 polypeptides are also provided. Methods for the use of these novel sequences to inhibit expression or function of cytochrome P450 polypeptides involved in this metabolic conversion are also provided. The methods find use in the production of tobacco products that have reduced levels of nornicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products.

First claim

What is claimed is:

1. A tobacco seed or a tobacco plant grown therefrom, wherein said tobacco seed or tobacco plant comprises a stable mutation in a wild type allele encoding a nicotine demethylase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 4, wherein said mutation reduces or eliminates the expression or activity of said nicotine demethylase, wherein the amount of nornicotine or N′-nitrosonornicotine in said tobacco plant is reduced by greater than about 10% compared to a control tobacco plant comprising said wild type allele, wherein said tobacco plant is a Nicotiana tabacum plant.

2. The tobacco seed or tobacco plant grown therefrom according to claim 1 , wherein said tobacco seed or tobacco plant grown therefrom is homozygous for said mutation.

3. The tobacco seed or tobacco plant grown therefrom according to claim 1 , wherein said tobacco plant has a non-converter phenotype and reduced nicotine demethylase activity from said nicotine demethylase.

4. The tobacco seed or tobacco plant grown therefrom according to claim 1 , wherein said tobacco plant has a non-converter phenotype and comprises less than 5% nicotine demethylation.

5. The tobacco seed or tobacco plant grown therefrom according to claim 1 , wherein said tobacco seed or plant grown therefrom is selected from the group consisting of a Burley type, a dark type, a flue-cured type, and an Oriental type.

6. The tobacco seed or tobacco plant grown therefrom according to claim 1 , wherein said mutation is selected from the group consisting of a point mutation, a deletion, and an insertion.

7. The tobacco seed or tobacco plant grown therefrom according to claim 1 , wherein said wild type allele encodes SEQ ID NO: 4.

8. The tobacco seed or tobacco plant grown therefrom according to claim 1 , wherein the amount of nornicotine or N′-nitrosonornicotine in said tobacco plant is reduced by greater than about 20% compared to a control tobacco plant comprising said wild type allele.

9. The tobacco seed or tobacco plant grown therefrom according to claim 1 , wherein the amount of nornicotine or N′-nitrosonornicotine in said tobacco plant is reduced by greater than about 60% compared to a control tobacco plant comprising said wild type allele.

10. The tobacco seed or tobacco plant grown therefrom according to claim 1 , wherein the amount of nornicotine or N′-nitrosonornicotine in said tobacco plant is reduced by greater than about 90% compared to a control tobacco plant comprising said wild type allele.

11. A method comprising preparing a tobacco product using a cured tobacco leaf from the tobacco plant of claim 1 .

12. The method of claim 11 , wherein said tobacco product is selected from the group consisting of a cigarette, a cigar, pipe tobacco, snuff, chewing tobacco, gum, and a lozenge.

13. A tobacco product comprising cured tobacco from the tobacco plant of claim 1 , wherein said tobacco product is selected from the group consisting of a cigarette, a cigar, pipe tobacco, snuff, chewing tobacco, gum, and a lozenge.

14. A method comprising:
a. detecting a mutation in a tobacco plant or plant part thereof, wherein said mutation is at a locus encoding a polypeptide having at least 96% sequence identity to SEQ ID NO: 4, and wherein said mutation is in a sequence corresponding to the codon sequence encoding the tryptophan residue at position 329 of SEQ ID NO: 4; and
b. producing a tobacco product from said tobacco plant or plant part thereof comprising said mutation detected in step a.

15. The method of claim 14 , wherein said method further comprises isolating a nucleic acid molecule from said tobacco plant, said plant part thereof, or said tobacco product; and
detecting said mutation in said nucleic acid molecule.

16. The method of claim 14 , wherein said tobacco plant is a Nicotiana tabacum plant.

17. The method of claim 14 , wherein said tobacco plant is selected from the group consisting of a Burley type, a dark type, a flue-cured type, and an Oriental type.

18. The method of claim 14 , wherein said plant part is selected from the group consisting of a cell, a protoplast, a cell tissue culture from which a whole plant can be regenerated, a callus, an embryo, pollen, an anther, an ovule, a seed, a leaf, a flower, a stem, a branch, a fruit, a root, and a root tip.

19. The method of claim 14 , wherein said tobacco product is selected from the group consisting of a cigarette, a cigar, pipe tobacco, snuff, chewing tobacco, gum, and a lozenge.

20. The method of claim 14 , wherein said mutation reduces or eliminates the expression or activity of said polypeptide.

Assignees

• Univ North Carolina State
• Univ Kentucky Res Found

Inventors

• Dewey Ralph E
• Siminszky Balazs
• Bowen Steven W
• Gavilano Lily

Classifications

• A24B15/10

  Chemical features of tobacco products or tobacco substitutes · CPC title

• C12Q1/6895

  for plants, fungi or algae · CPC title

• A01H6/823

  Nicotiana, e.g. tobacco · CPC title

• C12N9/0073

  with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13 · CPC title

• A24B13/00

  Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff (reconstituted tobacco products A24B3/14) · CPC title