红紫芽茶高花青素相关GT家族基因克隆与功能研究

Anthocyanidins are the main reddishviolet substances in plants, and has strong healthcare effects on oxidation resistance and anti-inflammatory. Reddishviolet tea plants (Camellia sinensis) are considered as a new type of tea owning ornamental and healthcare characteristics simultaneously, with tender shoots presenting bright red or purple perennially because of high content anthocyanidins in these tissues. Glycosytransferases (GTs) are the key enzymes in anthocyanidin synthesis, catalyzing the glycosylation of anthocyanidins and enhancing their stability. However, up to now, little is known about the molecular base of high content anthocyanidins in these resources. Previously, we had bred a batch new lines of reddishviolet tea plants, established the differential genes expression profiles between reddishviolet leaves and green ones, and cloned three ESTs encoding new GTs in tea plant, which were dramatically up-regulated in reddishviolet leaves. In this study, we will focus on the isolation and functional characterization of the three GT genes, including cloning of full-length cDNA and promoter sequences, gene function investigation in vivo and enzyme activity detection in vitro by transforming tea plant calli, screening the upstream transcription regulators using yeast one-hybrid assay, analyzing the correlation between GT genes expression and anthocyanidins accumulation in tea leaves temporally and spatially, confirmation the roles of GTs in anthocyanidins synthesis in tea plants. The implementation of this project will contribute to illuminate the molecular mechanism of high content anthocyanidins in reddishviolet tea plants, as well as develop novel tea products with high content anthocyanidins, which surely has good application prospects.

花青素是植物红紫色主要呈色物质，具有抗氧化、消炎等多种保健功能。红紫芽茶树因其新梢芽叶富含花青素而常年呈鲜艳的红色或紫色，是兼具保健和观赏价值的新型特色茶。糖基转移酶（GT）是花青素合成的关键酶，负责催化花青素的糖基化，提高花青素的稳定性。目前，针对茶树花青素合成相关GT家族基因的研究还鲜有报导。前期，项目组选育了一批红紫芽茶树新品系，通过构建红紫叶与绿叶差异基因表达谱，获得了3个GT新基因的EST，这些基因在红紫叶中的表达量显著高于绿叶。本项目拟在此基础上重点对这3个GT基因进行全长cDNA和启动子克隆、转化茶树愈伤组织进行体内功能研究和体外酶活鉴定、酵母单杂交获得调控这些基因的转录因子，并分析其表达与茶叶花青素积累的时空进程关系，明确GTs在茶树花青素合成中的作用。项目实施不仅对阐明红紫芽茶树高花青素形成的分子机理具有重要意义，还将为研制新型高花青素茶叶产品提供指导，应用前景良好。

花青素是植物红紫色主要呈色物质，具有抗氧化、消炎等多种保健功能。红紫芽茶树因其新梢芽叶富含花青素而常年呈鲜艳的红色或紫色，是兼具保健和观赏价值的新型特色茶。糖基转移酶（GT）是花青素合成的关键酶，负责催化花青素的糖基化，提高花青素的稳定性。目前，针对茶树花青素合成相关GT家族基因的研究还鲜有报导。前期，项目组选育了一批红紫芽茶树新品系，通过构建红紫叶与绿叶差异基因表达谱，获得了3个GT新基因的EST。本项目在此基础上，利用RT-PCR技术筛从中选出1个在不同红紫芽茶树品种红紫叶中的表达量均要高于绿叶的GT基因，半乳糖苷转移酶基因（CsF3GalT），并采用RACE技术克隆了其cDNA全长序列，生物信息学分析表明其ORF全长为1400 bp， 编码 454个氨基酸，其蛋白的分子大小约为 129.97 kDa，等电点为4.76；亚细胞定位发现CsF3GalT定位在细胞质、细胞膜和细胞核中；通过构建其超表达载体并经农杆菌介导转化拟南芥，发现转基因植株叶柄明显颜色加深变紫，对其上游Myb调控因子CsRAB转化拟南芥也发现，种子萌发后6天时，转基因幼苗茎部呈紫色，且两个基因在不同红紫芽茶品种和不同部位叶片的表达趋势一致，表明CsF3GalT和CsRAB是茶树花青素合成的重要参与因子；体外酶活实验证明，CsF3GalT蛋白为半乳糖基转移酶，而非葡萄糖基转移酶，且同一季节不同品种CsF3GalT活性与茶多酚总量和花青素含量均显著正相关；进一步的代谢组学分析表明，红紫芽品种鲜叶中矮牵牛-3-O-半乳糖的含量显著大于绿芽品种对照，90mg/ml浓度的半乳糖处理红紫芽品种红妃，锦葵色素-3-O-半乳糖苷含量显著上调，由此印证了CsF3GalT在茶树花青素的合成过程中可能的重要作用。由此，本研究基本明确了CsF3GalT在茶树花青素合成中的作用，这不仅对阐明红紫芽茶树高花青素形成的分子机理具有重要意义，还将为高花青素茶树资源快速鉴评、新品种选育和新型高花青素茶叶产品开发提供指导，应用前景良好。