摘要
黄草乌为云南的道地药材,其富含滇乌碱等二萜生物碱,二萜生物碱是其主要活性成分。法呢基焦磷酸合酶(FPS)是萜类生物合成途径中的一个关键酶,在二萜生物碱生物合成中发挥重要作用,其功能研究有助于揭示二萜生物碱合成的分子机制。该研究基于黄草乌转录组数据,筛选到1条FPS基因(AvFPS),克隆其全长序列并进行了生物信息学分析、功能验证以及基因表达分析。AvFPS开放阅读框(ORF)为1056 bp,编码351个氨基酸,相对分子质量约为41 kDa,其具有异戊烯基转移酶的2个典型保守功能域“DDIMD”和“DDYXD”。在大肠杆菌中表达AvFPS重组蛋白,用纯化重组蛋白进行体外酶促反应,结果表明,AvFPS能够催化法呢基焦磷酸(farnesyl pyrophosphate,FPP)的合成。qRT-PCR分析结果表明,AvFPS基因在黄草乌根、茎、叶和花中均有表达,在根中表达量最高;经MeJA诱导后,AvFPS的表达量明显上调。该研究明确了AvFPS的催化功能,揭示了AvFPS在不同组织以及经MeJA诱导不同时间段的表达模式,为更深入了解FPS基因在二萜类成分生物合成途径中的功能提供了参考。
Aconitum vilmorinianum is an authentic and superior medicinal herbal in Yunnan,which is rich in yunaconitine and other diterpene alkaloids.Diterpene alkaloids are its main active components.Farnesyl pyrophosphate synthase(FPS)is a key enzyme in the terpene biosynthetic pathway and plays an important role in diterpene alkaloid biosynthesis.Functional studies of FPS help to reveal the molecular mechanism of diterpene alkaloid biosynthesis.In this study,one FPS gene(AvFPS)was selected based on the transcriptome data of A.vilmorinianum.Its full-length sequence was cloned,and bioinformatic analysis,functional verification,and gene expression analysis were performed.The open reading frame(ORF)of AvFPS was 1056 bp,encoding 351 amino acids.Its molecular weight was 41 kDa.AvFPS had two typical conserved functional domains of isopentenyl transferase,"DDIMD"and"DDYXD".The recombinant protein of AvFPS was expressed in Escherichia coli,and purified recombinant protein was used for in vitro enzymatic reaction.The results revealed that AvFPS was able to catalyze the synthesis of farnesyl pyrophosphate(FPP).The results of qRT-PCR analysis showed that AvFPS was expressed in the roots,stems,leaves,and flowers of A.vilmorinianum,with the highest expression level in the roots.The expression level of AvFPS was significantly up-regulated by MeJA induction.This study clarified the catalytic function of AvFPS,revealed the expression pattern of AvFPS in different tissue,as well as at different time induced by MeJA,and provided a reference for a deeper understanding of the function of FPS in the biosynthesis of diterpenoid components.
作者
王雪
李国栋
王宝婕
徐福荣
马晓惠
WANG Xue;LI Guo-dong;WANG Bao-jie;XU Fu-rong;MA Xiao-hui(Key Laboratory of Yunnan Provincial Department of Education on Substance Benchmark Research of Ethnic Medicines,Yunnan University of Chinese Medicine,Kunming 650500,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2024年第16期4387-4395,共9页
China Journal of Chinese Materia Medica
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项目(202305AF150018)
中央本级重大增减支项目(2060302)
云南省“高层次人才培养支持计划”青年拔尖人才项目(YNWR-QNBJ-2020-278)。
关键词
黄草乌
法呢基焦磷酸合酶
功能验证
表达分析
Aconitum vilmorinianum
farnesyl pyrophosphate synthase
functional verification
expression analysis
