川西獐牙菜SmGES基因的克隆、生物信息学分析与原核表达  

Cloning, Bioinformatics Analysis and Prokaryotic Expression of SmGES Gene in Swertia mussotii

作　　者：李晓雪[1] 孙继奇 王天宇 田丰收 张程 朱晔荣[1] 向蓓蓓[2] 王勇[1] Li Xiaoxue;Sun Jiqi;Wang Tianyu;Tian Fengshou;Zhang Cheng;Zhu Yerong;Xiang Beibei;Wang Yong(College of Life Science,Nankai University,Tianjin 300071;School of Chinese Materia Medica,Tianjin University of Traditional Chinese Medicine,Tianjin 301617)

机构地区：[1]南开大学生命科学学院,天津300071 [2]天津中医药大学中药学院,天津301617

出　　处：《中国农学通报》2020年第25期19-25,共7页Chinese Agricultural Science Bulletin

基　　金：天津市自然科学基金“藏药川西獐牙菜呫吨酮类化合物合成生物学关键元件的初步研究”(18JCQNJC14000);国家自然科学基金“川西獐牙菜环烯醚萜合成酶的克隆和功能验证”(81303303)。

摘　　要：研究藏药川西獐牙菜中环烯醚萜合成途径中的关键酶香叶醇合成酶的功能,并探讨其在香叶醇、龙胆苦苷和獐牙菜苦苷生物合成中的作用。以川西獐牙菜叶片为材料,提取RNA并反转为c DNA,通过川西獐牙菜转录组相关信息,用RT-PCR技术克隆得到了川西獐牙菜香叶醇合成酶基因,并命名为SmGES基因。将SmGES基因片段通过分子生物学的方法连接到原核表达载体pET-28a上并转化到Escherichia coli Rosetta(DE3)原核表达感受态中,进行相关的诱导表达。通过生物信息学进行相关分析得出,SmGES基因长1761 bp,编码586个氨基酸;SmGES蛋白等电点为5.35,相对分子质量为67.78 kDa。分析表明SmGES基因带有叶绿体转运肽,预测其是定位于叶绿体的亲水蛋白。文章还分析了该蛋白的细胞内定位、结构域、二级和三级结构。进化关系分析表明,SmGES基因与滇龙胆的GES基因具有较近的亲缘关系。诱导得到重组蛋白分子质量67.78 kDa的蛋白条带,与预期大小一致。研究结果为进一步阐明川西獐牙菜中环烯醚萜合成途径提供参考。The paper aims to study the function of the key enzyme geraniol synthase in the terpenoids pathway in Swertia mussotii, a tibetan medicine, and to explore its role in the biosynthesis of geraniol, swertiamarin and gentianaceae. RNA was extracted from leaves of S. mussotii and converted into cDNA. According to the transcriptom of S. mussotii, we cloned the geraniol synthase of S. mussotii by RT-PCR and named SmGES gene. The SmGES gene fragment was connected to the prokaryotic expression vector p ET-28 a and transformed into the Escherichia coli Rosetta(DE3) for induction expression. According to bioinformatics analysis, the length of SmGES gene was 1761 bp and 586 amino acids were encoded. The isoelectric point of SmGES protein was 5.35 and the relative molecular weight was 67.78 kDa. Analysis showed that SmGES gene contained chloroplast transporter, which was predicted to be a hydrophilic protein located in chloroplast. The intracellular localization, structural domains, secondary and tertiary structures of the protein were analyzed. The results showed that SmGES gene was closely related to GES gene of Gentinan rigescens. The recombinant protein with a molecular weight of 67.78 kDa was induced, which was consistent with the expected size. The study could lay a foundation for further elucidate the synthetic pathway of terpenoids in the S. mussotii.

关 键 词：川西獐牙菜 香叶醇合成酶基因 基因克隆 生物信息学 原核表达 

分 类 号：Q946.5[生物学—植物学]