青皮苦瓜鲨烯合酶基因ORF序列的克隆和正选择分析  

Cloning and Positive Selection Analysis of Squalene Synthase Gene ORF in Green Momordica charantia

作　　者：钱洁颖 马成通 晁耐霞[1,2] 陈奇聪 蓝秀万[1,2] 孙健[1,2] 吴耀生[1,2] 

机构地区：[1]广西医科大学生物化学与分子生物学教研室,南宁530021 [2]广西高校生物分子医学研究重点实验室,南宁530021

出　　处：《基因组学与应用生物学》2016年第8期2114-2124,共11页Genomics and Applied Biology

基　　金：国家自然科学基金(No.31260069)资助

摘　　要：为研究葫芦科植物中三萜皂苷生物合成通路中的关键酶鲨烯合酶的分子进化,克隆青皮苦瓜鲨烯合酶基因的c DNA序列,并进行正选择分析。根据葫芦科植物之间的同源性设计青皮苦瓜鲨烯合酶引物。采用3'RACE和5'RACE快速扩增技术分两步进行巢式PCR扩增鲨烯合酶ORF序列。根据克隆出来的青皮苦瓜编码框序列以及在Gen Bank数据库中完整的陆生植物鲨烯合酶编码框序列信息,用贝叶斯方法和PAML4软件进行鲨烯合酶的正选择分析。青皮苦瓜鲨烯合酶基因c DNA的克隆,命名为Mc SS,其c DNA序列全长为1 548 bp,ORF 1 251 bp,编码417个氨基酸残基。通过正选择运算,检测到33个正选择位点,其正选择位点主要集中在第二跨膜区及其两侧。本研究成功克隆得到青皮苦瓜鲨烯合酶基因全长c DNA序列并对其正选择分析,在三萜合成通路上为鲨烯合酶的定点突变提供重要参考。To explore the molecular evolution and characteristics of SS in Cucurbitacine biosynthesis pathway of Cucurbitaceae, we cloned the eDNA of key enzyme squalene synthase from Green Moraordica charantia, and made analysis of positive selection. Primers of squalene synthase of Green Momordica charantia were designed to amplify 3＇-terminal and 5＇-terminal sequence of SS by 3＇-RACE and 5＇-RACE respectively, based on Cucurbitaceae Gynostemmapentaphyllum, Siraitia grosvenorii, Trichosanthes rubriflos squalene synthase with 3＇ RACE and 5＇ RACE rapid amplification of PCR amplification to obtain the eDNA. With the sequences published in GenBank, we use Bayesian methods and PAML4 to conduct positive selection analysis. Green Momordica charantia squalene synthase eDNA named as McSS and gained full-length sequence （1 548 bp）, ORF 1 251 bp, encoding 417 amino acid residues. According to the positive selection operation, we got 33 positively selected sites. Surprisedly, most positively selected sites centralized on the second transmembrane domain. We successfully cloned the full length eDNA sequence of Green Momordica charantia squalene synthase gene and did positive selected analysis, providing an important reference for squalene synthase site-directed mutagenesis involved in triterpenoid saponins biosynthesis pathway.

关 键 词：青皮苦瓜 基因克隆 鲨烯合酶 正选择 

分 类 号：S642.5[农业科学—蔬菜学] Q943.2[农业科学—园艺学]