Reducing levels of nicotinic alkaloids in plants
View Patent ↗Two genes, A622 and NBB1, can be influenced to achieve a decrease of nicotinic alkaloid levels in plants. In particular, suppression of one or both of A622 and NBB1 may be used to decrease nicotine in tobacco plants.
1. A method of producing a reduced-nicotine tobacco plant, comprising introducing an isolated nucleic acid encoding SEQ ID NO: 4 or a fragment thereof of at least 20 contiguous nucleotides to genetically engineer suppression of a gene product encoded by SEQ ID NO: 3 in said plant.
2. A reduced-nicotine tobacco plant produced by the method of claim 1 .
3. The method of claim 1 , wherein the engineering comprises introducing into a plant cell of said plant a nucleic acid construct that expresses a short interfering RNA that suppresses expression of a gene product encoded by SEQ ID NO: 3.
4. The method of claim 1 , wherein the engineering comprises introducing into a plant cell of said plant a nucleic acid construct, comprising, in the 5′ to 3′ direction, a promoter operably linked to a heterologous nucleic acid encoding SEQ ID NO: 3 in sense or antisense orientation, and a terminator.
5. A cDNA molecule comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence as set forth in SEQ ID NO: 3; and
(b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4.
6. A genetically engineered tobacco plant comprising a chimeric nucleic acid construct comprising said nucleic acid molecule of claim 5 linked to a heterologous nucleic acid.
7. Seeds from said plant of claim 6 , wherein said seeds comprise said nucleic acid construct.
8. A reduced-nicotine tobacco product comprising said plant of claim 6 .
9. A method for reducing an alkaloid in a tobacco plant, comprising introducing the nucleic acid molecule of claim 5 to genetically engineer suppression of a gene product encoded by SEQ ID NO: 3.
10. The method of claim 9 , wherein said alkaloid is anatabine.
11. A genetically engineered tobacco plant having suppression of a gene product encoded by SEQ ID NO: 3, wherein said plant exhibits reduced expression of said gene product compared to a non-transformed control and said plant comprises plant cells containing:
a nucleic acid construct comprising in the 5′ to 3′ direction, a promoter operable in said plant cell and a heterologous DNA operably associated with said promoter, wherein said heterologous DNA comprises the DNA sequence of SEQ ID NO: 3.
12. The genetically engineered tobacco plant of claim 11 , wherein said heterologous DNA is in antisense orientation.
13. The genetically engineered tobacco plant of claim 11 , wherein said heterologous DNA is in the sense orientation.
14. Progeny of the plant according to claim 11 , wherein said progeny have suppression of a gene product encoded by SEQ ID NO: 3.
15. Seeds of the plant according to claim 11 , wherein said seeds comprise said nucleic acid construct.
16. A method of making a genetically engineered reduced-nicotine tobacco cell having suppression of a gene product encoded by SEQ ID NO: 3, said method comprising:
(a) providing a plant cell of a type known to express said gene product;
(b) providing a nucleic acid construct, comprising, in the 5′ to 3′ direction, a promoter operable in a plant cell and a nucleic acid sequence encoding an mRNA for said gene product, said nucleic acid operably associated with said promoter; and
(c) transforming said plant cell with said nucleic acid construct o produce transformed cells having suppression of said gene product.
17. A plant cell produced by the method of claim 3 , 4 , or 16 .
18. A method for reducing an alkaloid in a tobacco plant, comprising:
(a) introducing the isolated nucleic acid of claim 5 to genetically engineer suppression of a gene product encoded by SEQ ID NO: 3; and
(b) genetically engineering suppression of at least one additional nicotine biosynthesis enzyme selected from the group consisting of aspartate oxidase, quinolinate synthase, quinolate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyltransferase, diamine oxidase, and A622.
19. The method of claim 18 , wherein said alkaloid is anatabine and a gene product encoded by SEQ ID NO: 3 and putrescine N-methyltransferase are suppressed.
20. A genetically engineered tobacco plant produced by the method of claim 19 , wherein said plant is characterized by decreased anatabine, and suppression of a gene product encoded by SEQ ID NO: 3 and putrescine N-methyltransferase.
21. The method of claim 18 , wherein said alkaloid is anatabine, and a gene product encoded by SEQ ID NO: 3, A622, and putrescine N-methyltransferase are suppressed.
22. A genetically engineered tobacco plant produced by the method of claim 21 , wherein said plant is characterized by decreased anatabine, and suppression of a gene product encoded by SEQ ID NO: 3, A622, and putrescine N-methyltransferase.
23. A method for reducing total alkaloid content in a tobacco plant comprising:
(a) introducing the isolated nucleic acid molecule of claim 5 to genetically engineer suppression of a gene product encoded by SEQ ID NO: 3; and
(b) genetically engineering suppression of at least one additional nicotine biosynthesis enzyme selected from the group consisting of aspartate oxidase, quinolinate synthase, quinolate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyltransferase, diamine oxidase, and A622.
24. The method of claim 23 , wherein said method comprises genetically engineering suppression of a gene product encoded by SEQ ID NO: 3 by introducing the isolated nucleic acid molecule of claim 5 and quinolate phosphoribosyl transferase.
25. A genetically engineered tobacco plant produced by the method of claim 24 , wherein said plant is characterized by decreased total alkaloid content, and genetically engineered suppression of a gene product encoded by SEQ ID NO: 3 and quinolate phosphoribosyl transferase.
26. A method for reducing total alkaloid content in a tobacco plant, comprising genetically engineering suppression of NBB1 by introducing the isolated nucleic acid molecule of claim 5 , and genetically engineering suppression of A622, and PMT.
27. A genetically engineered tobacco plant produced by the method of claim 26 , wherein said plant is characterized by decreased total alkaloid content, and suppressed NBB1 as recited in SEQ ID NO: 4, A622, and putrescine N-methyltransferase.
28. A method for reducing total alkaloid content in a tobacco plant, comprising genetically engineering suppression of a gene product encoded by SEQ ID NO: 3 by introducing the nucleic acid molecule of claim 5 , A622, and QPT.
29. A genetically engineered tobacco plant produced by the method of claim 28 , wherein said plant is characterized by decreased total alkaloid content, and suppression of a gene product encoded by SEQ ID NO: 3, A622, and quinolate phosphoribosyl transferase.
30. A tobacco plant having suppression of a gene product encoded by SEQ ID NO: 3 and reduced alkaloid content, wherein said suppression of said gene product and reduced total alkaloid content results from genetic engineering by introducing the isolated nucleic acid molecule of claim 5 .
31. A vector comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence as set forth in SEQ ID NO: 3; and
(b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4.
32. A recombinant nucleic acid molecule comprising a nucleotide sequence comprising SEQ ID NO: 3, operably linked to a heterologous nucleic acid sequence.
33. A probe or primer comprising SEQ ID NO: 3, wherein said probe or primer comprises a detectable label.