茶树茶氨酸合成酶基因的酶活性验证与蛋白三维结构分析

针对NCBI上已登录的茶氨酸合成酶与谷氨酰胺合成酶基因序列进行克隆、原核表达与酶活性验证,利用多种生物信息学数据库和软件,对Cs TS与Cs GS基因进行结构、性质和功能预测,采用同源建模法对蛋白三维结构进行预测,比较并预测催化作用位点的差异;用系统进化树分析从裸子植物到高等被子植物的谷氨酰胺合成酶基因序列,推测其进化的演变过程;通过对原核表达的基因工程菌提取粗酶液进行酶活性测定。结果表明:尽管茶树TS与GS序列高度同源,但是原核表达后的融合蛋白仍然显示了不同的催化能力,蛋白一、二级结构分析显示Cs TS与Cs GS差异不大,但是通过同源建模形成的蛋白三级结构分析显示,Cs TS与Cs GS存在3个催化位点上的差异,这可能是导致其酶活性差异的关键。系统进化分析结果首次确定茶氨酸合成酶应为谷氨酰胺合成酶基因家族成员,按照其细胞定位预测应为胞质型GS,其亲缘关系与同为双子叶植物的葡萄、陆地棉、巴西橡胶树、拟南芥较接近。

Enzymatic activity verification and protein three-dimensional structural analysis of theanine synthetase gene in Camellia sinensis

The aim of this study was to analyze the highly homologous sequences of theanine synthetase genes and glutamine synthetase genes of tea plant,speculate their evolutionary relationship,predict their structures,natures,functions and differences of enzyme active sites,and then to provide a basis for CsTS genes function studies of tea. Two gene sequences logged on NCBI were cloned,the expression of enzymes activities was verificated through HPLC and LC-MS and phylogenetic analysis was implemented within the searching result. The expression of enzymes in E. coli suggested CsTS and CsGS had different catalytic activities although their high homology. The primary and secondary protein structures of CsTS and CsGS were analyzed and the result showed that there was no significant difference between them, but the third protein structure analysis by homology modeling in the catalytic sites showed three differences in the catalytic sites, which could lead to the difference of enzyme activity. In this research, CsTS gene sequences were firstly identified as the members of GS gene family through phylogenetic analysis. According to cellular localization prediction, CsTS should belong to cytoplasmic GS(GS1), for its kinship close to grape, upland cotton, rubber tree, arabidopsis etc.