植物次生代谢产物生物合成转录调控是植物科学研究热点之一。托品烷类生物碱（TAs）生物合成途径功能基因和代谢工程研究很深入，但TAs生物合成转录调控研究还没有报道。 本研究拟开展WRKY类转录因子在TAs生物合成中的调控作用研究。以药典收录TAs药源植物颠茄为材料，以颠茄不同组织转录组测序结果和TAs途径功能基因启动子分析为基础，分离鉴定与TAs合成途径中PMT、TRI、CYP80F1和H6H等功能基因表达具有协同作用的WRKY；通过EMSA分析WRKY与PMT、TRI、CYP80F1和H6H启动子结合活性，采用酵母单杂交和瞬时表达分析WRKY对这4个基因启动子的激活活性；最后采用转基因方法在颠茄发根中实现WRKY的RNAi和Overexpression，研究转WRKY基因对TAs生物合成途径相关基因表达和对应代谢产物合成的影响，从而阐明WRKY转录因子调控TAs生物合成的分子机理。

Transcritional regualtion of plant secondary metabolite biosynthetic pathways is a hot field of plant science. The functional genes involved in tropane alkaloid (TAs) biosynthesis have been intensively studied and metabolic engineering of TAs biosynthesis is very successful. Unfortunately, there are no reports on transcription factors regulating TAs biosynthesis. In the proposal, we will study WRKY transcription factors (TFs) regulating TAs biosynthesis. Atropa belladonna, the medicine-source plant of TAs authorized by Chinese Pharmacopoeia, is used as the plant material. We respectively isolated the promoter of PMT and H6H and found that both of them had the W box that was the binding site of WRKY TFs. The WRKY TFs, of which expression are coordinate with the functional genes involved in TAs pathway such as PMT, CYP80F1, TRI and H6H, are the candidate TFs controling TAs biosynthesis. The candidate WRKY TFs can be isolated based on the sequenced transcritomes of different organs of A. belladonna and expression profile analysis of WRKY and the four TAs-pathway functional genes. Then EMSA will be used to test the binding of WRKY to the W boxs in the promoters of PMT, CYP80F1, TRI and H6H; yeast one-hybird will be applied to investigate the activition of the promoters of the four genes by WRKY. Finally, the WRKY TFs will be decreased by RNAi or be increased by overexpression at the transcritional levels in A. belladonna via transgenic technology; and expression of genes involved in TAs biosynthetic pathway and corresponding metabolites will be analyzed in transgenic A. belladonna (WRKY RNAi / Overexpression). All the researches above will explain molecular mechanism of WRKY TFs regulating biosynthesis of TAs.