Nucleic acid sequences encoding transcription factors regulating alkaloid biosynthesis and their use in modifying plant metabolism
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.