Identification of Negative Regulators Involving in Nicotine Biosynthesis in Tobacco

Nicotine is the predominant alkaloid in tobacco (Nicotiana tobacum) and determines smoke quality and causing addiction to smoking. Nicotine biosynthesis is regulated by jasmonic acid (JA) signaling transduction pathway. MYC2, as a functionally conserved factor, regulates downstream gene expressions directly or indirectly. Previously, our results demonstrated that CrMYC2 interacts with transcriptional repressor CrGBF to antagonic inhibit terpenoid indole alkaloid biosynthesis in Catharanthus roseus. Similarly, we also found that overexpression of NtMYC2 also triggered the antagonic inhibition on nicotine biosynthesis gene expression in tobacco. In this proposal, we will conduct transcriptomic analysis on NtMYC2 overexpression and RNAi transgenic lines and orthological comparison of other transcriptional repressor sequence to investigate candidate transcription factor function as a the NtMYC2 midaited negative regulator in nicotine biosynthesis. Phylogenetic analysis, subcellular localization, DNA-protein (or protein-protein) interaction assay, nicotine biosynthetic gene expression levels and nicotine contents in transgenic overexpression and knock down plants will be analyzed to verify the inhibition function of candidate regulator. The results will not only further our understanding of the inhibitory regulation of nicotine biosynthesis, but also provide gene resource which can be used in tobacco breeding program