中药材2024，Vol.47Issue(9)：2198-2202,5.DOI:10.13863/j.issn1001-4454.2024.09.010
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
摘要
Abstract
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 1 215 bp and 1 206 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 type Ⅱ thiolytase.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Key words
Aconitum vilmorinianum Kom./Acetyl-CoA C-acetyltransferase/Gene cloning/Real-time fluorescence quantification-polymerase chain reaction/Prokaryotic expression分类
中医学引用本文复制引用
王雪,曹小青,郭冬梅,徐福荣,马晓惠..黄草乌乙酰辅酶A酰基转移酶基因的克隆与表达分析[J].中药材,2024,47(9):2198-2202,5.基金项目
云南省应用基础中医联合专项项目(202101AZ070001-053) （202101AZ070001-053）
云南省王源超专家工作站(202305AF150018) （202305AF150018）
中央本级重大增减支项目(2060302) （2060302）
云南省科技人才和平台计划项目(202105AG070012). （202105AG070012）
