茶树单宁酶基因的挖掘及功能验证

Galloylated catechins in the tea plant are the main phenolic compounds that determine the astringency taste of teas. In our previous study, we found that the content of galloylated catechins was affected by the reaction of synthesis, condensation, and hydrolysis in tea plant [Camellia sinensis (L.) O. Kuntze]. Meanwhile, we found that more than one tannase in tea plant could hydrolyze the gallate ester bond of hydrolytic tannins and galloylated catechins. Up to now, the genes encoding tannase have not been fully clear. In this project, in order to excavate those genes involved in the hydrolysis of gallate ester bond of hydrolytic tannins or condensed tannins, total tannase in the tea plant will firstly been isolated and purified by ammonium sulfate precipitation, hydrophobic interaction chromatography (HIC), concanavalin A (ConA) chromatography, and gel filtration. The purified proteins will been identified by MALDI-TOF-MS/MS, and the candidate genes will been screened based on the mass spectrometric datas from tea plant genome and transcriptome databases. The heterogeneous expression of candidate genes in E. coli and purification of recombinant tannase will been performed, and biochemical characteristics of the recombinant enzyme will been analyzed in vitro. Then, the relationship between the structure and catalytic mechanism of different tannase proteins will been analyzed by enzyme kinetics, crystal structure analysis and point mutation technique. Furthermore, the physiological functions of different tannase genes will been evaluated by using the established transgenic system, including instantaneous expression of tea plant. Excavation of different tannase genes in the tea plant has an important scientific significance and valuable application to develop tea varieties with low astringency and to screen the excellent germplasm resources from tea plants, grapes and persimmons.

酯型儿茶素是决定茶叶涩味的主要酚类化合物。我们前期研究表明，茶树中酯型儿茶素含量受到合成、缩合和水解代谢的综合影响；并发现茶树中至少存在一种以上的单宁酶类可水解酯型儿茶素和水解单宁中的没食子酯键。迄今为止，编码单宁酶的基因尚没有被挖掘出来。本项目首先以茶树鲜叶为材料，采取“单宁酶蛋白纯化-蛋白肽链的MALDI-TOF-MS/MS分析-转录组基因组数据搜索候选基因-重组蛋白表达及酶活检测”技术，筛选出茶树不同单宁酶基因；通过各个重组蛋白的酶动力学性质、晶体结构解析和点突变技术，解析不同单宁酶蛋白结构和催化机理之间关系；利用已建立的包括茶树瞬时表达在内的转基因体系，评估不同单宁酶基因的生理功能。挖掘茶树不同单宁酶基因，对于开发低涩味茶树品种，筛选茶树及葡萄、柿子等优良种质资源均具有重要的科学意义和应用价值。

没食子酰基化儿茶素是决定茶叶涩味的主要酚类化合物。我们前期研究表明，茶树中酯型儿茶素含量受到合成、缩合和水解代谢的综合影响；并发现茶树中至少存在一种以上的单宁酶类可水解酯型儿茶素和水解单宁中的没食子酯键。本项目利用“蛋白质纯化-蛋白质肽链的MALDI-TOF -MS/MS分析-转录组数据搜索-基因克隆表达分析及功能验证”的方法，筛选并验证到了一个植物的单宁酶基因家族。这是首次报道植物的单宁酶基因家族。并发现植物单宁酶是一组同时具有水解酶和转酰基酶活性的混杂酶。利用草莓瞬时遗传转化体系，研究了草莓单宁酶基因对草莓品质的影响；建立了茶树和杨梅树的瞬时转基因表达体系，并利用这个体系验证单宁酶在茶树和杨梅树种酚类代谢中的作用。遗传转化结果表明植物单宁酶基因具有促进没食子衍生物合成的功能而不是促进它们的降解。本项培养1名博士后、4名博士生和4名硕士生。提供学生参加2次国际会议并做大会报告。课题迄今为止，已经在New Phytologist、Plant J、Food Chem和JAFC等知名国际杂志上发表标记文章十多篇。申请专利2项。挖掘茶树不同单宁酶基因，对于开发低涩味茶树品种，筛选茶树及葡萄、柿子等优良种质资源均具有重要的科学意义和应用价值。

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