UDP-糖基转移酶参与茶树中苦涩味类黄酮双糖苷的生物合成研究

茶树中多类型且高含量的类黄酮糖苷，特别是类黄酮双糖苷，赋予了茶叶特殊的收敛性口感和保健功效。然而，目前该类化合物在茶树中的合成机理尚不清楚。研究表明，UDP-糖基转移酶(UGTs)蛋白家族参与了上述类黄酮糖苷的合成。前期我们虽然从茶树中鉴定了4条参与类黄酮单糖苷合成的UGTs基因，但是迄今为止参与类黄酮双糖苷合成的UGTs还没有找到。因此，本项目拟通过代谢组学与酶活力关联分析，筛选类黄酮双糖苷高积累且合成能力强的茶鲜叶材料，利用该材料进行“植物源蛋白纯化→蛋白质测序→基因筛选”实验，挖掘参与类黄酮双糖苷合成的UGTs；其次，利用原核表达及酶活性分析，研究UGTs的体外催化功能；最后，利用拟南芥遗传转化体系和基因沉默技术，研究上述UGTs的表达与植物中类黄酮双糖苷的积累关系。本项目旨在通过研究茶树中类黄酮双糖苷生物合成的分子机理，为低涩味茶树品种选育和类黄酮糖苷药物的开发等提供理论依据。

The flavonoid glycosides with multi-type and high level in tea plant, especially flavonoid diglycosides, give tea beverage with special astringency taste and pharmacological benefits on human health. However, the mechanism of the biosynthesis of these compounds in tea plant is still unclear. It is generally believed that UDP-glycosyltransferase (UGTs) are responsible for catalyzing the glycosidation of flavonoids in plants. Indeed, in our previous studies, we have identified and verified 4 UGTs involved in the biosynthesis of flavonoid monoglucoside from tea plant. However, due to the lack of effective experimental methods, the UGTs involved in the synthesis of flavonoid diglycosides have not been found so far. Based on the above problems, this project puts forward a technical proposal: firstly, the fresh tea leaves with high accumulation and strong synthesis ability of flavonoid diglycosides were selected by the correlation analysis of metabonomics and enzyme activity. After that, the target UGTs that involved in the synthesis of flavonoid diglycosides can be identified by means of applying the techniques of "purification of plant protein → protein sequencing → gene screening" with the materials mentioned above. Secondly, the function of these target UGTs was studied by prokaryotic expression and enzyme activity analysis in vitro. Finally, the relationship between the expression of UGTs and the accumulation of flavonoid diglycosides in plants was confirmed by using Arabidopsis genetic transformation system and gene silencing technology. This project aims to provide theoretical basis for the selection of low astringency tea varieties and the drugs development of flavonoid glycosides by studying the biosynthesis and its molecular mechanism of flavonoid diglycosides in tea plants.