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. A method for increasing a nicotinic alkaloid in a Nicotiana plant, comprising growing the plant under conditions which allow for the expression of a transcription factor from an exogenous nucleic acid sequence in the plant, wherein the exogenous nucleic acid sequence is introduced using a vector comprising a nucleotide sequence selected from the group consisting of:
(i) the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6;
(ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7;
(iii) a nucleotide sequence that is at least 90% identical to the nucleotide sequences of (i) or (ii), and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis; and
(iv) a nucleotide sequence that hybridizes under high stringency conditions to the complement of the nucleotide sequences of (i), (ii), and/or (iii), and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis;
wherein expression of the transcription factor results in the plant having an increased nicotinic alkaloid content as compared to a control plant grown under similar conditions.
2. The method of claim 1 , wherein the plant overexpresses at least one of NBB1, A622, quinolate phospho-ribosyltransferase (QPT), putrescine-N-methyltransferase (PMT), and N-methylputrescine oxidase (MPO).
3. The method of claim 1 , wherein the vector comprises the nucleotide sequence set forth in SEQ ID NO: 5.
4. The method of claim 1 , wherein the vector comprises the nucleotide sequence set forth in SEQ ID NO: 6.
5. The method of claim 1 , wherein the vector comprises a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7.
6. The method of claim 1 , wherein the vector comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 5, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.
7. The method of claim 1 , wherein the vector comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 6, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.
8. The method of claim 1 , wherein the vector comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.
9. The method of claim 1 , wherein the vector comprises a nucleotide sequence which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis and which hybridizes under high stringency conditions to the complement of a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence set forth in SEQ ID NO: 5;
(b) a nucleotide sequence set forth in SEQ ID NO: 6;
(c) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7;
(d) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 5, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis;
(e) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 6, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis; and
(f) a nucleotide sequence that is at least 90% identical to a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.
10. The method of claim 2 , wherein NBB1 is additionally up-regulated.
11. The method of claim 2 , wherein A622 is additionally up-regulated.
12. The method of claim 2 , wherein QPT is additionally up-regulated.
13. The method of claim 2 , wherein PMT is additionally up-regulated.
14. The method of claim 2 , wherein MPO is additionally up-regulated.
15. The method of claim 1 , wherein the plant overexpresses at least one additional transcription factor that positively regulates nicotinic alkaloid biosynthesis.
16. The method of claim 15 , wherein the vector comprises the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6.
17. The method of claim 15 , wherein the vector comprises a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7.
18. The method of claim 15 , wherein the vector comprises a nucleotide sequence which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis and which is at least 90% identical to the nucleotide sequence of (a) SEQ ID NO: 5, (b) SEQ ID NO: 6, or (c) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7.
19. The method of claim 15 , wherein the vector comprises a nucleotide sequence which encodes a transcription factor that positively regulates alkaloid biosynthesis and which hybridizes under high stringency conditions to the complement of a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 5;
(b) the nucleotide sequence set forth in SEQ ID NO: 6;
(c) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7;
(d) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 5, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis;
(e) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 6, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis; and
(f) a nucleotide sequence that is at least 90% identical to a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.
20. The method of claim 1 , wherein the plant exhibits suppression of at least one additional transcription factor that negatively regulates nicotinic alkaloid biosynthesis.
21. The method of claim 20 , wherein the vector comprises the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6.
22. The method of claim 20 , wherein the vector comprises a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7.
23. The method of claim 20 , wherein the vector comprises a nucleotide sequence which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis and which is at least 90% identical to the nucleotide sequence of (a) SEQ ID NO: 5, (b) SEQ ID NO: 6, or (c) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7.
24. The method of claim 20 , wherein the vector comprises a nucleotide sequence which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis and which hybridizes under high stringency conditions to the complement of a nucleotide sequence selected from the group consisting of
(a) the nucleotide sequence set forth in SEQ ID NO: 5;
(b) the nucleotide sequence set forth in SEQ ID NO: 6;
(c) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7;
(d) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 5, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis;
(e) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 6, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis; and
(f) a nucleotide sequence that is at least 90% identical to a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.