茶树儿茶素没食子酰基化关键基因SCPL4转录调控研究

酯型儿茶素是茶树最重要的次生代谢成分。我们前期发现并鉴定了儿茶素没食子酰基化的关键基因和合成途径，但对其转录调控研究尚属空白。本项目拟开展儿茶素没食子酰基化关键基因启动子元件功能分析和相关合成调控转录因子筛选及鉴定，旨在为调控夏秋茶苦涩味、挖掘优质茶树基因资源、开发优质茶树品种奠定理论基础。本项目主要研究内容：（1）克隆儿茶素没食子酰基化关键基因CsSCPL4的启动子序列，构建携带报告基因载体，转化模式植物验证启动元件功能；（2）基于已有茶树cDNA酵母筛选文库，利用酵母单杂技术，结合数据库分析、转录组-多酚代谢组关联分析，筛选与上述启动子结合的转录因子；（3）利用茶树瞬时表达体系，结合EMSA和LUC实验，进一步确认参与茶树儿茶素没食子酰基化的转录因子；（4）利用酵母双杂，结合Co-IP-MS、BiFC和Pull-Down技术，筛选并鉴定参与转录因子调控的互作蛋白及其互作机理。

Galloylated catechins are the most important second matebolites in tea plants. The applicant's previous studies identified the crucial step of catechin galloylation and the key genes in tea plants. However, research on its transcription-level regulation is till blank. This research focus on analysis of the promoter element functions, screening and identification of assistant transcriptional factor involved in catechin galloylation. These studies will establish a solid theoretical foundation for regulation the bitterness and astringency of summer- and autumn- tea leaves, exploiting high-quality genetic resources in tea plants, developing high-quality varieties of tea plants. The main researches includes: (1) Cloning promoters of key genes CsSCPL4, constructing vectors merged with reporter gene, and then transfering into model plants to verify promoter element functions; (2) Based on the established cDNA library of tea plant, screening the transcription factors binding to the above promoters by integrating yeast one-hybrid screen with related database analysis and correlation analysis of transcriptome- polyphenol metabolome; (3) Identifying the transcription factors by combing transient system of tea plants with EMSA and LUC technologies; (4) Screening and verifying the interacting proteins involved in transcriptional regulation, and investigating the interaction mechanism among proteins by combining yeast two-hybrid system with Co-IP-MS, BiFC and pull-down technologies.