黄草乌乙酰辅酶A酰基转移酶基因的克隆与表达分析  

Cloning and Expression Analysis of Acetyl-CoA Acetyltransferase Gene from Aconitum vilmorinianum

作　　者：王雪 曹小青 郭冬梅 徐福荣 马晓惠 WANG Xue;CAO Xiao-qing;GUO Dong-mei;XU Fu-rong;MA Xiao-hui(Yunnan University of Chinese Medicine/Key Laboratory of Yunnan Provincial Department of Education on Substance Benchmark Research of Ethnic Medicines,Kunming 650500,China;School of Traditional Chinese Medicine,Yunnan University of Chinese Medicine/Yunnan Key Laboratory of Sustainable Utilization of Southern Medicine,Kunming 650500,China)

机构地区：[1]云南中医药大学/云南省教育厅民族药物质基准研究重点实验室,云南昆明650500 [2]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：《中药材》2024年第9期2198-2202,共5页Journal of Chinese Medicinal Materials

基　　金：云南省应用基础中医联合专项项目(202101AZ070001-053);云南省王源超专家工作站(202305AF150018);中央本级重大增减支项目(2060302);云南省科技人才和平台计划项目(202105AG070012)。

摘　　要：目的:克隆并分析黄草乌乙酰辅酶A酰基转移酶(AACT)基因,以便解析黄草乌中二萜生物碱的合成调控机制。方法:基于黄草乌转录组数据筛选并克隆AvAACT基因;应用相关软件对其进行生物信息学分析;在大肠杆菌中表达该基因的编码蛋白,并利用实时荧光定量PCR检测AvAACT基因在植物体各组织及MeJA诱导后的表达情况。结果:从黄草乌中克隆获得2条AvAACT基因,AvAACT1和AvAACT2的开放阅读框分别为1 215 bp和1 206 bp,分别编码404个和401个氨基酸。生物信息学分析显示,该氨基酸序列与唐松草等植物中的AACT有较高的同源性,都具有Ⅱ型硫解酶催化作用的活性中心结构域。在大肠杆菌中成功表达了AvAACTs重组蛋白。qRT-PCR分析结果表明,AvAACTs基因在黄草乌根、茎、叶和花中均有表达,AvAACT1在花中表达量最高,AvAACT2在叶中表达量最高;经MeJA诱导后,黄草乌AvAACTs的表达量均明显上调。结论:该研究从黄草乌中克隆出AvAACTs基因的全长序列,并在大肠杆菌中成功表达其重组蛋白,同时明确了黄草乌不同组织以及经MeJA处理不同时间后该基因的表达模式,为进一步挖掘AvAACTs基因的功能积累了坚实的基础。Objective:To clone and analyze the gene of acetyl-CoA acyltransferase(AACT)in Aconitum vilmorinianum,so as to elucidate the mechanism of diterpenoid alkaloid synthesis in Aconitum vilmorinianum.Methods:The AvAACT gene was screened and cloned based on the transcriptome data of Aconitum vilmorinianum.The bioinformatics analysis was carried out by using related soft-ware.The encoded protein of AvAACT gene was expressed in Escherichia coli,and the expression of AvAACT gene in the tissues of the plants and MeJA induction was detected by real-time fluorescence quantitative PCR.Results:Two AvAACT genes were cloned from Aco-nitum vilmorinianum.The open reading frames of AvAACT1 and AvAACT2 were 1215 bp and 1206 bp,encoding 404 and 401 amino acids,respectively.Bioinformatic analysis showed that the amino acid sequences had high homology with AACT in plants such as Thalic-trum thalictroides,and both of them had active center domains catalyzed by typeⅡthiolytase.The recombinant protein AvAACTs was successfully expressed in Escherichia coli.The results of qRT-PCR showed that AvAACTs gene were expressed in roots,stems,leaves and flowers of Aconitum vilmorinianum,and the expression of AvAACT1 was the highest in flowers and the expression of AvAACT2 was the highest in leaves.After induction by MeJA,the expression of AvAACTs in Aconitum vilmorinianum was significantly up-regulated.Con-clusion:In this study,the full-length sequence of AvAACTs gene is cloned from Aconitum vilmorinianum,and its recombinant protein is successfully expressed in Escherichia coli.Meanwhile,the expression patterns of AvAACTs gene in different tissues of Aconitum vilmorin-ianum and at different time after induction by MeJA are compared,which accumulate a solid foundation for further exploring the function of AvAACTs gene.

关 键 词：黄草乌 乙酰辅酶A酰基转移酶 基因克隆 实时荧光定量-聚合酶链式反应 原核表达 

分 类 号：R282.71[医药卫生—中药学]