Nucleic acid sequences encoding transcription factors regulating alkaloid biosynthesis and their use in modifying plant metabolism
View Patent ↗Plant metabolism and alkaloid levels can be regulated by transcription factors that regulate the nicotinic alkaloid biosynthetic pathway. In one embodiment, the disclosure provides a transcription factor that negatively regulates alkaloid biosynthesis, such as nicotine biosynthesis.
1. An isolated cDNA molecule comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9;
(b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10; and
(c) a nucleotide sequence that is at least 90% identical to the nucleotide sequences of (a) or (b);
wherein (a), (b), and (c) encode a transcription factor that positively regulates nicotinic alkaloid biosynthesis.
2. A Nicotiana cell comprising the cDNA molecule of claim 1 .
3. The Nicotiana cell of claim 2 , wherein the level of the transcription factor in the Nicotiana cell is higher than in a Nicotiana cell that does not contain the nucleic acid molecule.
4. An expression vector comprising the nucleic acid molecule of claim 1 , operably linked to one or more control sequences suitable for directing expression in a Nicotiana cell.
5. A Nicotiana cell comprising the expression vector of claim 4 .
6. The Nicotiana cell of claim 5 , wherein the level of the transcription factor in the Nicotiana cell is higher than in a Nicotiana cell that does not contain the expression vector.
7. The isolated cDNA molecule of claim 1 , wherein the nucleotide sequence is set forth in SEQ ID NO: 8.
8. The isolated cDNA molecule of claim 1 , wherein the nucleotide sequence is set forth in SEQ ID NO: 9.
9. The isolated cDNA molecule of claim 1 , wherein the nucleotide sequence is at least 90% identical to the nucleotide sequence of SEQ ID NO: 8, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.
10. The isolated cDNA molecule of claim 1 , wherein the nucleotide sequence is at least 90% identical to the nucleotide sequence of SEQ ID NO: 9, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.
11. The isolated cDNA molecule of claim 1 , wherein the nucleotide sequence is at least 90% identical to the nucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.
12. A method for reducing a nicotinic alkaloid in a Nicotiana plant, comprising down-regulating a transcription factor that positively regulates nicotinic alkaloid biosynthesis, wherein the transcription factor is down-regulated by:
(a) genetically engineering a population of plant cells to eliminate production of a protein encoded by the nucleotide sequence set forth in SEQ ID NO: 8, comprising introducing one or more mutations into the region of SEQ ID NO: 8 that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10; and
(b) detecting and selecting a target mutated plant cell or a plant derived from such a cell, wherein the target mutated plant cell or plant has a mutation in a gene encoding a transcription factor that positively regulates nicotinic alkaloid biosynthesis and wherein the targeted plant cell or plant has a decreased nicotinic alkaloid content relative to a control plant cell or plant.
13. The method of claim 12 , wherein the genetic engineering further comprises introducing into the population of cells a reagent comprising a recombinagenic oligonucleobase.
14. The method of claim 12 , wherein the genetic engineering further comprises introducing into the population of cells a reagent comprising a targeted nuclease.
15. The method of claim 12 , wherein the genetic engineering further comprises suppressing within the Nicotiana plant at least one of NBB1, A622, quinolate phospho-ribosyltransferase (QPT), putrescine-N-methyltransferase (PMT), and N-methylputrescine oxidase (MPO).
16. The method of claim 12 , wherein the genetic engineering further comprises suppressing within the Nicotiana plant at least one additional transcription factor that positively regulates nicotinic alkaloid biosynthesis.
17. The method of claim 12 , wherein the genetic engineering further comprises overexpressing within the Nicotiana plant at least one transcription factor that negatively regulates nicotinic alkaloid biosynthesis.
18. The method of claim 12 , wherein the genetic engineering further comprises introducing one or more mutations into regions of SEQ ID NO: 8 resulting in a complete deletion of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 10.
19. A mutated Nicotiana plant produced by the method of claim 12 , wherein the plant has reduced expression of a transcription factor that positively regulates nicotinic alkaloid biosynthesis and reduced nicotinic alkaloid content, as compared to a control plant.
20. The mutated plant of claim 19 , wherein the plant is a Nicotiana tabacum plant.
21. A product having a reduced level of a nicotinic alkaloid comprising the mutated plant of claim 19 , or portions thereof, relative to a product produced from a control plant.
22. The product of claim 21 , wherein the product is selected from the group consisting of a cigarette, cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewing tobacco, snuff, snus, lozenges, and reconstituted tobacco.
23. Seeds from the mutated plant of claim 19 .