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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。
Abstract：To explore the molecular evolution and characteristics of SS in Cucurbitacine biosynthesis pathway of Cucurbitaceae, we cloned the c DNA of key enzyme squalene synthase from Green Momordica charantia, and made analysis of positive selection. Primers of squalene synthase of Green Momordica charantia were designed to amplify3'-terminal and 5'-terminal sequence of SS by 3'-RACE and 5'-RACE respectively, based on Cucurbitaceae Gynostemma pentaphyllum, Siraitia grosvenorii, Trichosanthes rubriflos squalene synthase with 3' RACE and 5' RACE rapid amplification of PCR amplification to obtain the c DNA. With the sequences published in Gen Bank, we use Bayesian methods and PAML4 to conduct positive selection analysis. Green Momordica charantia squalene synthase c DNA named as Mc SS 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 c DNA 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.
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基本信息:
DOI：10.13417/j.gab.035.002114
中图分类号:S642.5;Q943.2
引用信息:
[1]钱洁颖,马成通,晁耐霞,等.青皮苦瓜鲨烯合酶基因ORF序列的克隆和正选择分析[J].基因组学与应用生物学,2016,35(08):2114-2124.DOI:10.13417/j.gab.035.002114.
基金信息:
国家自然科学基金(No.31260069)资助
2016-08-25
2016-08-25