翼首草鲨烯合酶(PhSQS2)全长克隆、表达特性分析及蛋白功能验证  

Cloning,expression pattern and functional analysis of squalene synthase(PhSQS2)in Pterocephali Herba

作　　者：王芸[1] 吴宇 蒋庆锋 何焱 兰小中[4] 张磊[1,2] WANG Yun;WU Yu;JIANG Qing-feng;HE Yan;LAN Xiao-zhong;ZHANG Lei(Biomedical Innovation R&D Center,School of Medicine,Shanghai University,Shanghai 200444,China;Department of Pharmaceutical Botany,School of Pharmacy,Second Military Medical University,Shanghai 200433,China;Pharmacy Department,Medical Security Center,General Hospital of Tibet Military Region,Lhasa 850000,China;Animal Husbandry College and TAAHC-SWU Medicinal Plant R&D Centre,Tibet Agricultural and Animal Husbandry University,Nyingchi 860000,China)

机构地区：[1]上海大学医学院生物医药创新研发中心,上海200444 [2]海军军医大学药学院药用植物学教研室,上海200433 [3]西藏军区总医院医疗保障中心药剂科,西藏拉萨850000 [4]西藏农牧学院西南大学药用植物联合研发中心,西藏林芝860000

出　　处：《中草药》2022年第21期6840-6847,共8页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(31970316);上海市优秀学术带头人计划(19XD1405000)。

摘　　要：目的从匙叶翼首草Pterocephalus hookeri转录组中挖掘参与三萜合成的鲨烯合酶(squalene synthase,SQS)并研究其作用机制。方法以翼首草基原植物匙叶翼首草为研究对象,基于前期转录组数据筛选克隆PhSQS2基因,并进行生物信息学分析;通过烟草瞬时转化实验观察PhSQS2亚细胞定位情况,利用实时荧光定量PCR分析PhSQS2在不同器官中的表达特征;构建原核表达载体经体外酶促反应鉴定PhSQS2功能。结果PhSQS2基因开放阅读框(open reading frame,ORF)区域全长1242 bp编码414个氨基酸,蛋白相对分子质量为47400,理论等电点为6.57,总平均疏水性为-0.075,为不稳定蛋白。PhSQS2主要定位在细胞核和细胞质中;组织分布具有特异性,在根和叶中均有表达,叶中表达量显著高于根中。体外酶促实验证明PhSQS2催化法尼基焦磷酸生成鲨烯。结论PhSQS2的功能鉴定为解析翼首草中萜类物质生物合成途径,提高三萜皂苷类成分含量及优质种质资源筛选培育提供了前期理论基础。Objective To explore the squalene synthase(SQS) from the transcriptome of Pterocephalus hookeri and investigate its mechanism of action. Methods Taking P. hookeri as research object, the PhSQS2 gene was cloned and bioinformatically analyzed based on preliminary transcriptome data;The subcellular localization of PhSQS2 was observed by transient transformation experiments in tobacco, and the expression characteristics of PhSQS2 in different organs were analyzed by real-time fluorescence quantitative PCR;Finally, the prokaryotic expression vector was constructed to identify the function of PhSQS2 by enzymatic reaction in vitro. Results The PhSQS2 gene was screened with the length of open reading frames 1242 bp, encoding proteins 414amino acids. Bioinformatics analysis showed that it was an unstable hydrophobic protein with a relative molecular weight of 47 400,a theoretical PI of 6.57 and an average hydrophobicity of-0.075. PhSQS2 was mainly localized in nucleus and cytoplasm;Its tissue distribution was specific, and it was expressed in both roots and leaves, and the expression in leaves were significantly higher than those in roots. In vitro enzymatic reactions proved that as a squalene synthase, PhSQS2 catalysed the production of squalene from farnesyl pyrophosphate. Conclusion The functional identification of PhSQS2 provides a theoretical basis for the further analysis of the terpenoid biosynthesis pathway and the improvement of triterpenoid saponins from P. hookeri, as well as for in-depth research on the screening and cultivation of high-quality germplasm resources.

关 键 词：匙叶翼首草 翼首草 鲨烯合酶(PhSQS2) 生物信息学 表达特性 基因功能 

分 类 号：R286.12[医药卫生—中药学]