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: 5 or SEQ ID NO: 6;
(b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7; 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 alkaloid biosynthesis, and wherein the cDNA molecule is operably linked to one or more heterologous control sequences suitable for directing expression in a Nicotiana cell.
2. An expression vector comprising the cDNA molecule of claim 1 .
3. A Nicotiana cell comprising the expression vector of claim 2 .
4. The isolated cDNA molecule of claim 1 , wherein the nucleotide sequence is set forth in SEQ ID NO: 5.
5. The isolated cDNA molecule of claim 1 , wherein the nucleotide sequence is set forth in SEQ ID NO: 6.
6. The isolated cDNA molecule of claim 1 , wherein the nucleotide sequence is at least 90% identical to the nucleotide sequence of SEQ ID NO: 5, and which encodes a transcription factor that positively regulates nicotine biosynthesis.
7. The isolated cDNA molecule of claim 1 , wherein the nucleotide sequence is at least 90% identical to the nucleotide sequence of SEQ ID NO: 6, and which encodes a transcription factor that positively regulates nicotine biosynthesis.
8. 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: 7, and which encodes a transcription factor that positively regulates nicotine biosynthesis.
9. A method for reducing nicotine in a Nicotiana plant, comprising down-regulating a transcription factor that positively regulates nicotine biosynthesis, wherein the transcription factor is down-regulated by:
(a) introducing into a Nicotiana plant cell a nucleic acid comprising at least 21 consecutive nucleotides of a cDNA molecule of claim 1 , wherein the consecutive nucleotides are in sense orientation, antisense orientation, or both;
(b) producing a Nicotiana plant comprising the plant cell;
(c) growing the Nicotiana plant under conditions whereby the nucleotide sequence decreases levels of the transcription factor in the Nicotiana plant as compared to a control Nicotiana plant grown under similar conditions, and
(d) selecting a Nicotiana plant having normal development and/or physiology and reduced nicotine as compared to a non-transformed control Nicotiana plant.
10. The method of claim 9 , further comprising suppressing within the Nicotiana plant at least one of NBB1, A622, quinolate phospho-ribosyltransferase (QPT), putrescine-N-methyltransferase (PMT), and N-methylputrescine oxidase (MPO).
11. The method of claim 9 , further comprising suppressing the expression of at least one additional transcription factor that positively regulates nicotine biosynthesis.
12. The method of claim 9 , further comprising overexpressing at least one transcription factor that negatively regulates nicotine biosynthesis.
13. A method for reducing nicotine in a Nicotiana plant, comprising down-regulating a transcription factor that positively regulates nicotine biosynthesis, wherein the transcription factor is down-regulated by:
(a) introducing into a population of Nicotiana plant cells a reagent for site-directed mutagenesis of a target comprising at least 21 consecutive nucleotides of the cDNA molecule of claim 1 ; and
(b) detecting and selecting a target mutated Nicotiana plant cell or a Nicotiana plant derived from the Nicotiana cell, wherein the target mutated Nicotiana plant cell or Nicotiana plant has a mutation in a gene encoding a transcription factor positively regulating nicotine biosynthesis and reduced nicotine content as compared to a control Nicotiana cell or plant, and wherein the target mutated Nicotiana plant cell or Nicotiana plant exhibits normal development and/or physiology.
14. The method of claim 13 , wherein the reagent is a recombinagenic oligonucleobase.
15. The method of claim 13 , wherein the reagent is a targeted nuclease.
16. A mutated Nicotiana plant produced by the method of claim 13 , wherein the Nicotiana plant has reduced expression of a transcription factor that positively regulates nicotine biosynthesis and reduced nicotine content, as compared to a control Nicotiana plant.
17. A product comprising the mutated Nicotiana plant of claim 16 or portions thereof, wherein the product has a reduced level of nicotine as compared to a product produced from a control Nicotiana plant.
18. Seeds from the mutated Nicotiana plant of claim 16 .
19. A method for reducing nicotine levels in a population of Nicotiana plants, comprising:
(a) providing a population of mutated Nicotiana plants;
(b) detecting and selecting a target mutated Nicotiana plant exhibiting normal development and/or physiology within the population, wherein the target mutated Nicotiana plant has decreased expression of a transcription factor that positively regulates nicotine biosynthesis as compared to a control Nicotiana plant, wherein the detection comprises using a cDNA molecule of claim 1 , as primer or probe; and
(c) selectively breeding the target mutated Nicotiana plant to produce a population of Nicotiana plants having decreased expression of a transcription factor that positively regulates nicotine biosynthesis as compared to a population of control Nicotiana plants.