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.
1. A method for increasing 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 within said population, wherein said detecting comprises:
(i) using primers developed from SEQ ID NO: 14 to amplify regions of a transcription factor gene from mutated Nicotiana plants in the population of mutated Nicotiana plants;
(ii) identifying one or more mismatch between the amplified regions and corresponding regions in a wild-type gene, wherein the wild-type gene is from a non-mutated Nicotiana plant, and encodes a transcription factor that negatively regulates nicotine biosynthesis having an amino acid sequence that is at least 90% identical to SEQ ID NO: 16; and
(iii) identifying the mutated Nicotiana plant that contains the one or more mismatch, wherein said identified target mutated Nicotiana plant has decreased expression of a transcription factor that negatively regulates nicotine biosynthesis and increased nicotine levels, compared to a control Nicotiana plant comprising the wild type gene; and
(c) selectively breeding the target mutated Nicotiana plant and producing a population of plants having decreased expression of the transcription factor that negatively regulates nicotine biosynthesis and increased nicotine levels, compared to a population of control plants.
2. The method of claim 1 , wherein said plant is N. tabacum.
3. A Nicotiana plant having increased nicotine levels produced by the method of claim 1 .
4. A product comprising nicotine, said product produced from a harvested portion of the Nicotiana plant of claim 3 , wherein said product has an increased level of nicotine compared to a product produced from a wild-type Nicotiana plant of the same species.
5. A tissue culture comprising the plant cells of the Nicotiana plant of claim 3 , wherein said culture has increased production or secretion of nicotine.
6. A method for producing nicotine, comprising isolating nicotine from the tissue culture of claim 5 .
7. The tissue culture of claim 5 , wherein said culture has increased production or secretion of nicotine of at least 10% compared to culture comprising wild-type Nicotiana plant cells.