山茱萸萜类甲羟戊酸合成途径关键酶CoHMGS基因的克隆与分析  

Cloning and analysis of a key enzyme of CoHMGS gene involved in mevalonate pathway of terpene biosynthesis in Cornus officinalis

作　　者：侯典云[1,2] 王瑶瑶 刘晓冉 张佳琪 马占强 胥华伟[1,2] 王笑尘 HOU Dian-yun;WANG Yao-yao;LIU Xiao-ran;ZHANG Jia-qi;MA Zhan-qiang;XU Hua-wei;WANG Xiao-chen(College of Tree Peony,Henan University of Science and Technology,Luoyang 471023,China;The Luoyang Engineering Research Center of Breeding and Utilization of Dao-di Herbs,Luoyang 471023,China;Nan yang Institute of Cornus officinalis,Nanyang 474550,China)

机构地区：[1]河南科技大学农学院,河南洛阳471023 [2]洛阳市道地药材繁育与创新利用工程技术研究中心,河南洛阳471023 [3]南阳市山茱萸研究所,河南南阳474550

出　　处：《中草药》2021年第18期5716-5721,共6页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(U1404829);中央本级重大增减支项目“名贵中药资源可持续利用能力建设项目”(2060302);河南省自然科学基金项目(202300410151);河南省科技攻关项目(202102110156)。

摘　　要：目的克隆得到山茱萸Cornus officinalis萜类合成途径的关键酶CoHMGS基因的cDNA序列,并进行相应的生物信息学分析,为深入研究CoHMGS基因的功能奠定基础。方法以山茱萸转录组筛选出的c102453_g2序列为参考序列设计特异引物,以山茱萸叶片总RNA,通过RT-PCR扩增获得CoHMGS基因序列,序列纯化回收后连接到pTOPO-T载体上,转化至大肠杆菌DH10B,选取阳性克隆测序。利用生物信息学软件预测CoHMGS基因及其编码蛋白质的功能。结果克隆得到长度为1645 bp的CoHMGS序列,包含1413 bp的完整开放阅读框,编码470个氨基酸。蛋白整体带负电荷,为不稳定亲水性蛋白,定位于细胞质内,不含跨膜结构。结论首次克隆得到山茱萸CoHMGS基因,对其编码蛋白质进行了初步的分析和预测,为深入揭示CoHMGS基因在山茱萸萜类物质合成途径中的功能奠定了基础。Objective To clone the cDNA sequence of the key enzyme CoHMGS gene in the terpene synthesis pathway of Cornus officinalis and analyze its physical and chemical properties.Methods Specific primers were designed based on the>c102453_g2 sequence screened from the transcriptome.The template was the cDNA obtained by reverse transcription of the total RNA of leaves.The CoHMGS gene sequence was amplified by RT-PCR,and the amplified gene sequence was purified and recovered and then connected to the pTOPO-T vector,transforming into E.coli DH10B,and selecting positive clones for sequencing.The relevant bioinformatics software was used to predict the function of CoHMGS gene and its encoded protein.Results A 1645 bp CoHMGS sequence was cloned,containing a complete open reading frame of 1413 bp,encoding 470 amino acids.The protein was an unstable hydrophilic protein.It was predicted that its sub-cells were located in the cytoplasm,did not contain transmembrane structures,and were extra-membrane proteins.Conclusion The HMGS gene of C.officinalis was cloned for the first time,and a preliminary analysis and prediction of its encoded protein was made,which provided basic information for further revealing the synthesis pathway of terpenoids in C.officinalis.

关 键 词：山茱萸 甲羟戊酸途径 CoHMGS RT-PCR扩增 基因克隆 

分 类 号：R282.12[医药卫生—中药学]