茶树儿茶素没食子酰基化关键基因SCPL4转录调控研究

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.

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。

酯型儿茶素是茶树最重要的次生代谢成分。本课题是在前期明确了酯型（没食子酰基化）儿茶素合成途径的基础上，继续开展酯型儿茶素合成调控研究，鉴定调控酯型合成的转录因子。本课题开展了项目申请书的计划研究内容，并根据执行期间遇到的实际情况，开展了与本课题相关的研究内容。主要研究内容如下：（1）克隆酯型儿茶素合成关键基因CsSCPL4的启动子序列，开展了酵母单杂实验；（2）进一步完善了茶叶酚类代谢谱；（3）通过山茶属20种代表性植物鲜叶酚类代谢组和转录组的关联性分析，筛选获得一组调控茶树酯型儿茶素合成的CsMYBC2s转录因子。（4）利用双荧光素互补和酵母单杂等技术，验证了CsMYBC2s与CsTT8的互作关系，并研究了它们对酯型儿茶素合成关键基因CsLAR、CsANS、CsSCPL4及CsSCPL5的调控，表明CsMYBC2s在酯型儿茶素合成中发挥负调控的作用；（5）项目在执行过程中，发现CsSCPL5作为CsSCPL4的伴侣，共表达促进CsSCPL4蛋白翻译后加工，形成具有活性的酯型儿茶素合成酶ECGT。（6）项目在执行过程中，发现茶树鲜叶中的酯型儿茶素含量受到一种水解酶的动态调控。通过酶纯化结合质谱分析，以及基因功能验证，鉴定了该酶属于单宁酶。本研究从基因转录调控水平研究了茶酯型儿茶素合成酶的转录调控，从蛋白表达与酶学水平初步研究了酯型儿茶素合成酶形成和催化机制，从物质代谢水平研究了上下游酶类对酯型儿茶素积累的影响;取得了本领域多项原创性研究成果;为进一步研究植物代谢物酰基化修饰，以及茶品质调控奠定了坚实理论基础。