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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。
In order to study the molecular evolution of the key squalene synthase in the triterpene saponin biosynthesis pathway in Cucurbitaceae, the c DNA sequence of the squalene synthase gene was cloned and positively selected. According to the homology between the Cucurbitaceae plants, the green peel bitter squalene synthase primers were designed. The squalene synthase ORF sequence was amplified by nested PCR using 3’RACE and 5’RACE rapid amplification. According to the cloned sequence of green bitter melon and the complete terrestrial plant squalene synthase coding sequence information in Gen Bank database, the positive selection of squalene synthase was analyzed by Bayesian method and PAML4 software. The c DNA of crab squalene synthase gene was named Mc SS. The full-length c DNA sequence was 1 548 bp with an ORF of 1 251 bp, encoding 417 amino acid residues. Through positive selection operation, 33 positive selection sites were detected, and their positive selection sites mainly concentrated on the second transmembrane region and its two sides. In this study, the full-length c DNA sequence of squalene synthase gene was successfully cloned and its positive selection was analyzed. It provides an important reference for the site-directed mutagenesis of squalene synthase in the triterpene synthesis pathway.