茶树中茶氨酸水解酶分离纯化及其基因克隆研究

项目名称： 茶树中茶氨酸水解酶分离纯化及其基因克隆研究

项目编号： No.31200522

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 林学

项目作者： 李勤

作者单位： 湖南农业大学

项目金额： 23万元

中文摘要： 茶氨酸水解酶是茶树体内茶氨酸分解代谢的关键酶。但茶氨酸水解酶的分离纯化和基因克隆研究一直未取得突破性进展，严重制约了茶树体内茶氨酸代谢调控分子机制的深入研究。本项目拟以茶氨酸水解酶活性相对较高的茶鲜叶为材料，采用蛋白质层析系统和快速蛋白质纯化液相系统，通过多种蛋白质层析技术手段和酶活性保护剂的应用，分离纯化茶氨酸水解酶，并利用Edman降解法和MALDI-TOF MS/MS串联质谱对纯化的茶氨酸水解酶N-末端和内肽氨基酸序列进行分析；根据获得的部分氨基酸序列，通过RT-PCR和SMART RACE技术得到茶氨酸水解酶基因全长cDNA序列，为揭示茶树体内茶氨酸代谢调控分子机制及茶树种质资源改良奠定理论基础。

中文关键词： 茶氨酸水解酶；分离纯化；基因克隆；；

英文摘要： Theanine hydrolase is a key enzyme in the catabolism of theanine in Camellia sinensis. However, purification and cloning of theanine hydrolase has not been made a breakthrough progress, which significantly restrict the development of research on the molecular regulation of theanine metabolism in Camellis sinensis. In this project, high activity of theanine hydrolase of tea leaves are used as material, and theanine hydrolase is purified by several chromatography techniques and protector of enzyme on the protein purification system and fast protein liquid chromatography. The amino sequence of N-terminal and small peptide of theanine hydrolase is sequenced by Edman degradation and MALDI TOF MS/MS. According to the amino sequence of N-terminal and small peptide of theanine hydrolase, the full length cDNA of theanine hydrolase is obtained by RT-PCR and SMART RACE. This project provided a theoretical basis for the researchs on the molecular regulation of theanine mechanism and the germplasm improvement in Camellis sinensis.

英文关键词： theanine hydrolase；purify；gene clone；；