金铁锁鲨烯环氧酶基因的克隆与表达  

Cloning,Analysis and Expression of Squalene Epoxidase Gene in Psammosilene tunicoides

作　　者：李国栋[1] 韩丽君[1] 刘小莉[1] 杨耀文[1] 钱子刚[1] LI Guo-dong HAN Li-jun LIU Xiao-li YANG Yao-wen QIAN Zi-gang(Yunnan University of Traditional Chinese Medicine, Kunming 650500, Chin)

机构地区：[1]云南中医学院,云南昆明650500

出　　处：《中药材》2016年第10期2210-2216,共7页Journal of Chinese Medicinal Materials

基　　金：国家自然科学基金(81260609;81560613);云南省自然科学基金[2014FD035;2015FB205(-015)]

摘　　要：目的:从金铁锁Psammosilene tunicoides W.C.Wu et C.Y.Wu中克隆三萜皂苷合成途径中的关键酶鲨烯环氧酶(squalene epoxidase,SE)基因,并进行生物信息学和表达分析。方法:在转录组数据分析的基础上,利用RTPCR方法获得金铁锁SE基因的两个克隆的全长c DNA序列及进行生物信息学分析;并进行Real-time PCR实验,测定SE在不同组织中的表达量。结果:得到2条SE c DNA,SE-1全长1 642 bp,开放阅读框1 578 bp,编码525个氨基酸;SE-2全长1 552 bp,开放阅读框1 446 bp,编码481个氨基酸;构建了p EASY-E1-SE-1及p EASY-E1-SE-2重组质粒,获得稳定的原核表达体系,SDS-PAGE结果表明所表达的蛋白与预测的蛋白大小一致。Real-time PCR结果表明SE-1、SE-2基因在根中的表达量均高于茎和叶。结论:从金铁锁中成功克隆了SE基因,可为进一步研究金铁锁中三萜皂苷合成代谢途径及其关建酶表达模式奠定基础。Objective： To obtain the key enzyme gene squalene epoxidase（ SE） involving in the triterpene saponins biosynthesis,two SE gene were cloned from Psammosilene tunicoides,bioinformatics analysis and gene expression were performed. Methods： According to two SE gene sequences of transcriptome of Psammosilene tunicoides,two pairs of primers were designed,and the ORF（ Open reading frame） of c DNA sequences was obtained by RT-PCR. Then cloning and sequence analysis were performed. The expression profiles were validated across leaf,stem and root tissues through Real-time PCR. Results： The ORF of SE-1 had a length of 1 578 bp coding for 525 amino acids,and the ORF of SE-2 had a length of 1 446 bp coding for 481 amino acids. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size. The expression result showed that it was at much higher level in roots than in leaves and stems. Conclusion： The two SE genes were successfully cloned for the first time,and the stable prokaryotic expression system is established. This study will provide a foundation for the further purification,structural and functional research about triterpenoid biosynthesis of Psammosilene tunicoides.

关 键 词：金铁锁 鲨烯环氧酶 克隆 基因表达 生物信息学分析 

分 类 号：R978[医药卫生—药品]