UVR8介导的光信号途径调控茶树黄酮类合成机制研究

Flavonols are the most important secondary metabolites in tea (Camellia sinensis (L.) O. Kuntze) which determine the characteristics and health benefits of tea product. Exposure of the post-harvest fresh tea leaves to UV-B radiation, as one of the artificial manufacture process in tea production can improve the aroma and flavor characteristics of tea product through changing flavonol component. However, the UV-B signal transduction pathway and the role of UV-B in the regulation of flavonol biosynthesis in tea plants under nature environment remain unclear. Therefore, we constructed various UV environments for “Shuchazao” in one commercial tea plantation using different UV-transduction/excluding screens. The results demonstrated that UV-B induced changes in flavonols and gene expression of transcription factor CsHY5 which is involved in the UVR8-mediated signal transduction pathway and flavonol synthase CsFLS. The aim of this study is to analyze the role of UVR8-mediated signal transduction pathway in flavonol biosynthesis in tea plants under nature growth environment, especially the functions of CsHY5 and CsFLS through a range of technologies including the rapid-amplification of cDNA ends (RACE), subcellular localization by the green fluorescent protein (GFP), western blot and Arabidopsis uvr8 and hy5 complementation analysis, etc. We believe that this study provides valuable information to our understanding in light regulation of the secondary mechanism in the important commercial crop Camellia sinensis and the correlation between tea characteristics and its growth environments, also contributes to using UV-B radiation as one of the artificial processes to improve the quality of tea in industrial production.

黄酮类物质是决定茶叶品质特征和健康功效的主要茶树次生代谢产物之一。茶叶加工中使用UV-B辐射离体茶鲜叶能改变黄酮类组成，改善茶叶香气品质，然而UV-B在活体茶树内的信号转导与调控黄酮类合成分子机制尚不明确。为此，我们分别在茶园自然环境与人工气候室建立UV-B辐射茶树试验，发现UV-B能改变儿茶素含量并诱导光形态建成调控因子CsHY5与黄酮醇合成酶CsFLS表达，暗示CsHY5在茶树UV-B信号转导及调控黄酮类合成中发挥重要作用。本项目拟通过cDNA末端快速扩增、亚细胞定位、蛋白免疫印迹法及回补拟南芥突变体功能验证等，克隆茶树紫外光受体CsUVR8与CsHY5基因并检测其时空表达特征及在UV-B信号转导中的功能，解析茶园自然环境中UVR8介导的光信号途径调控茶树黄酮类合成的分子机制。本项目为诠释茶叶品质特征与茶区光照关系提供理论基础，对改进茶叶加工中利用UV-B提升茶叶品质也有积极作用。

黄酮类化合物是决定茶叶品质特征与健康功效的主要茶树次生代谢产物之一。茶叶加工中使用UV-B辐射离体茶鲜叶能改变黄酮类组成，改善茶叶香气品质，然而UV-B在活体茶树内的信号转导与调控黄酮类合成分子机制尚不明确。项目以国家级良种‘舒茶早’与黄化品种‘黄魁’为研究对象，建立茶园UV-B辐射茶树试验体系：首先，利用LC-MS技术鉴定茶树黄酮类物质对不同UV-B环境的响应，通过RNA-seq分析参与UV-B光信号转导、黄酮类物质合成相关基因与转录因子表达变化，并通过黄酮类物质含量-基因表达关联分析，靶标关键转录因子CsHY5与CsMYB12；其次，通过基因克隆、生物信息学分析与亚细胞定位等方法，分析CsHY5在茶树内表达特性；最后，通过酵母单双杂交、回补拟南芥hy5突变体功能验证等方法，解析CsHY5参与UV-B光信号调控茶树黄酮类物质合成的功能。研究发现UV-B光信号通过调控黄酮醇糖苷与儿茶素之间代谢流来影响茶叶苦涩味。滤除UV-B光组分，黄酮醇糖苷（尤其山奈酚-7-O-葡萄糖苷、杨梅素-3-O-葡萄糖苷与槲皮素-7-O-葡萄糖苷）含量减少，儿茶素类（儿茶素、没食子儿茶素和表没食子儿茶素）含量增加。参与UVR8光信号转导途径的关键转录因子（CsHY5、CsMYB12）与类黄酮生物合成途径关键基因（CsFLS、CsDFRa、CsLARa和CsMYB75）显著下调。进一步研究发现，UV-B光信号主要通过激活CsHY5-CsMYB12复合物表达，继而结合黄酮醇合成途径基因启动子（CsFLS、CsDFRa和CsLARa），调控黄酮类代谢物合成。此外，项目解析了遮荫条件下光信号调控叶绿体发育、叶绿素合成、改善茶叶色泽、提升茶叶口感的分子机理。本项目不仅为为诠释茶叶品质特征与茶区光照关系提供理论基础，也有助于优质茶、名优茶的生产，对改进茶叶加工中利用UV-B提升茶叶品质也有积极作用。