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 down-regulating a transcription factor that negatively regulates nicotinic alkaloid 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, wherein the target comprises at least 21 consecutive nucleotides of a nucleotide sequence selected from the group consisting of:
(i) a nucleotide sequence set forth in SEQ ID NO: 4; and
(ii) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of (i);
thereby producing a target mutated plant cell or a plant derived from such a cell; and
(b) detecting and selecting the target mutated Nicotiana plant cell or a Nicotiana plant, wherein the target mutated Nicotiana plant cell or Nicotiana plant has a mutation in a nucleotide sequence encoding the transcription factor and increased alkaloid content as compared to a control plant.
2. The method of claim 1 , wherein the reagent is a recombinagenic oligonucleobase.
3. The method of claim 1 , wherein the reagent is a targeted nuclease.
4. A mutated nicotinic alkaloid-producing Nicotiana plant produced by the method of claim 1 , wherein the Nicotiana plant has reduced expression of the transcription factor that negatively regulates nicotinic alkaloid biosynthesis and increased nicotinic alkaloid content as compared to a control Nicotiana plant grown under similar conditions.
5. The mutated Nicotiana plant of claim 4 , wherein the plant is a Nicotiana tabacum plant.
6. A product comprising the mutated Nicotiana plant of claim 4 or portions thereof, wherein the plant or portions thereof comprise the mutation in the nucleotide sequence encoding the transcription factor, and wherein the product has an increased level of nicotinic alkaloid as compared to a product comprising a control Nicotiana plant or portions thereof.
7. The product of claim 6 , 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, reconstituted tobacco, leaf tobacco, shredded tobacco, and cut tobacco.
8. Seeds from the mutated Nicotiana plant of claim 4 , wherein the seeds comprise the mutation in the nucleotide sequence encoding the transcription factor.