黄草乌异戊烯基焦磷酸异构酶基因的克隆与功能研究  

Cloning and functional characterization of isopentenyl diphosphate isomerase gene from Aconitum vilmorinianum

作　　者：马晓惠 曹小青 管丽娜 李国栋 王一博 MA Xiao-hui;CAO Xiao-qing;GUAN Li-na;LI Guo-dong;WANG Yi-bo(Yunnan Key Laboratory of Sustainable Utilization of Southern Medicine,College of Chinese Materia Medica,Yunnan University of Chinese Medicine,Kunming 650500,China)

机构地区：[1]云南中医药大学中药学院云南省南药可持续利用研究重点实验室,云南昆明650500

出　　处：《中草药》2022年第16期5142-5148,共7页Chinese Traditional and Herbal Drugs

基　　金：中央本级重大增减支项目(2060302);云南省科技人才和平台计划(202105AG070012)。

摘　　要：目的对黄草乌Aconitum vilmorinianum的异戊烯基焦磷酸异构酶(isopentenyl diphosphate isomerase,IDI)基因进行克隆及功能研究,为黄草乌中二萜生物碱的生物合成途径解析奠定基础。方法基于黄草乌的转录组数据筛选注释为IDI的基因,设计特异性引物克隆其编码区,利用相关软件对其进行生物信息学分析,利用实时荧光定量PCR分析其在黄草乌不同器官中的表达情况,在大肠杆菌中表达其重组蛋白,利用功能显色验证其功能。结果从黄草乌中克隆得到1个IDI基因(AvIDI,Genbank登录号MZ814967)。生物信息学分析表明,AvIDI的开放阅读框(open reading frame,ORF)为897 bp,编码298个氨基酸,分子式为C_(1516)H_(2362)N_(414)O_(452)S_(11),相对分子质量为33970,理论等电点为5.94,具有异戊烯基焦磷酸异构酶TNTCCSHPL和WGEHELDY 2个保守序列。系统进化分析显示,AvIDI与黄龙胆Gentiana lutea IDI的亲缘关系最近。表达分析表明,AvIDI基因在黄草乌根、茎、叶和花中均有表达,在茎中表达量最高。在大肠杆菌中成功表达了AvIDI重组蛋白,功能显色实验表明,AvIDI编码有功能的IDI蛋白,能促进大肠杆菌中番茄红素的积累。结论克隆得到AvIDI基因,并通过功能显色实验证明其编码有功能的IDI蛋白,该研究为利用IDI基因调控黄草乌中二萜生物碱生物合成奠定了基础。Objective To clone and functionally study the isopentenyl diphosphate isomerase(IDI)gene of Aconitum vilmorinianum,so as to lay a foundation for the analysis of the biosynthetic pathway of diterpene alkaloids in A.vilmorinianum.Methods The genes annotated as IDI were selected from the transcriptome data of A.vilmorinianum.Specific primers were designed to clone the coding regions.Bioinformatics analysis was performed with relevant software.Real-time quantitative PCR was used to analyze their expression level in different organs of A.vilmorinianum.The recombinant protein was expressed in Escherichia coli,and its function was verified by functional coloration experiment.Results An IDI gene(AvIDI,Genbank accession No.MZ814967)was cloned from A.vilmorinianum.Bioinformatics analysis showed that the open reading frame of AvIDI were 897 bp encoding 298 amino acids.The molecular formula of AvIDI was C_(1516)H_(2362)N_(414)O_(452)S_(11).The molecular weight of AvIDI was 33970 with a theoretical pI of 5.94.AvIDI contained two conserved sequences of TNTCCSHPL and WGEHELDY.Phylogenetic analysis showed that AvIDI was closely related to Gentiana lutea IDI.Expression analysis showed that AvIDI expressed in root,stem,leaf and flower of A.vilmorinianum,with highly expression in the stem.The recombinant protein of AvIDI was successfully expressed in E.coli.The functional coloration experiment in E.coli showed that AvIDI encoded a functional IDI protein and promoted the accumulation of lycopene.Conclusion The AvIDI was cloned and proven to encode a functional IDI protein.This study laid a foundation for the regulation of diterpenoid alkaloids biosynthesis in A.vilmorinianum with IDI gene.

关 键 词：黄草乌 异戊烯基焦磷酸酶 基因克隆 生物信息学分析 功能显色 

分 类 号：R282.12[医药卫生—中药学]