CAJ | 학술논문

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 CsTS 与CsGS 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 CsTS 与 CsGS 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,CsTS 与CsGS 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。
침대 NCBI 상이등록적다안산합성매여곡안선알합성매기인서렬진행극륭、원핵표체여매활성험증,이용다충생물신식학수거고화연건,대 CsTS 여CsGS 기인진행결구、성질화공능예측,채용동원건모법대단백삼유결구진행예측,비교병예측최화작용위점적차이；용계통진화수분석종라자식물도고등피자식물적곡안선알합성매기인서렬,추측기진화적연변과정；통과대원핵표체적기인공정균제취조매액진행매활성측정。결과표명：진관다수 TS 여 GS 서렬고도동원,단시원핵표체후적융합단백잉연현시료불동적최화능력,단백일、이급결구분석현시 CsTS 여 CsGS 차이불대,단시통과동원건모형성적단백삼급결구분석현시,CsTS 여CsGS 존재3개최화위점상적차이,저가능시도치기매활성차이적관건。계통진화분석결과수차학정다안산합성매응위곡안선알합성매기인가족성원,안조기세포정위예측응위포질형 GS,기친연관계여동위쌍자협식물적포도、륙지면、파서상효수、의남개교접근。
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 en-zymes in E .coli suggested CsTS and CsGS had different catalytic activities although their high homology.The pri-mary and secondary protein structures of CsTS and CsGS were analyzed and the result showed that there was no sig-nificant difference between them,but the third protein structure analysis by homology modeling in the catalytic sitesshowed 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.Accord-ing to cellular localization prediction,CsTS should belong to cytoplasmic GS (GS1),for its kinship close to grape, upland cotton,rubber tree,arabidopsis etc .