飞蝗核受体基因LmE75的分子特性和功能分析  

Molecular Characteristics and Function Analysis of Nuclear Receptor Gene LmE75 in Locusta migratoria

作　　者：刘晓健[1] 郭俊 张学尧[1] 马恩波[1] 张建珍[1] LIU XiaoJian;GUO Jun;ZHANG XueYao;MA EnBo;ZHANG JianZhen(Research Institute of Applied Biology,Shanxi University,Taiyuan 030006;College of Life Science,Shanxi University,Taiyuan 030006)

机构地区：[1]山西大学应用生物学研究所,太原030006 [2]山西大学生命科学学院,太原030006

出　　处：《中国农业科学》2020年第11期2219-2231,共13页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31402020,31872010);山西省高校科技创新项目(2016113);山西省面上青年基金项目(201601D021120)。

摘　　要：【目的】核受体(nuclear receptor,NR)是一类配体依赖型的转录因子,在昆虫生长发育过程中发挥重要作用,本文以重要农业害虫飞蝗(Locusta migratoria)为研究对象,分析核受体基因LmE75的分子特性以及功能,为害虫防治提供新分子靶标。【方法】基于飞蝗转录组数据库获得3条LmE75的cDNA序列,运用Blast和ExPASy网站中相关软件分析各个基因的开放阅读框、预测氨基酸序列、理化特性及保守结构域;使用MEGA 6.0软件中邻接法(neighbor-joining,NJ)与其他昆虫同源序列进行聚类分析。利用Primer 3.0软件设计3个LmE75亚型的特异性表达引物,运用reverse transcription-quantitative PCR(RT-qPCR)技术分析3个LmE75亚型在5龄若虫不同组织及4—5龄不同发育日龄的表达特性。体内注射蜕皮激素(20-hydroxyecdysone,20E)诱导和干扰20E受体基因LmEcR的表达后,RT-qPCR检测LmE75的表达,明确LmE75是否受20E调控。利用RNAi(RNA interference)技术,将体外合成的LmE75亚型共同区域的dsRNA注射至5龄飞蝗若虫体内,48 h后提取体壁RNA,反转录为cDNA,运用RT-qPCR技术检测LmE75亚型的沉默效率并观察表型;之后进一步利用H&E(hematoxylin and eosin)染色方法观察LmE75对飞蝗表皮结构的影响。【结果】基于飞蝗转录组数据库获得3条LmE75的cDNA序列,根据其结构特征命名为LmE75A、LmE75B和LmE75C,GenBank登录号分别为MN584732、MN584733和MN584734。3个LmE75亚型均具有典型的核受体结构域,其中DBD(DNA binding domain)和LBD(ligand binding domain)在各昆虫中高度保守;E75聚类分析结果显示不同目的昆虫类群E75亚型优先聚为一支;RT-qPCR结果显示3个LmE75亚型具有不同的组织和发育表达特性。LmE75A在体壁和肌肉中高表达;LmE75B在体壁中高表达,其次是前肠;LmE75C特异性在体壁高表达;LmE75A在4龄和5龄期均呈现先升高后降低的表达趋势;LmE75B在N4D3和N5D5表达量最高,蜕皮前后表达量均较低;LmE75C在4龄和5�【Objective】Nuclear receptors(NRs)act as ligand-inducible transcription factors,which play an important role during growth and development in insects.Locusta migratoria is an important agricultural pest.The objective of this study is to analyze the molecular characteristics and biological function of the nuclear receptor gene LmE75 in L.migratoria,and to provide a new molecular target for pest control.【Method】The cDNA sequences of three LmE75 genes were obtained based on the transcriptome database of L.migratoria.The open reading frames,predicted amino acid sequences,physicochemical properties and conserved domains of each gene were analyzed using Blast and ExPASy websites.Using MEGA 6.0 software,the neighbor-joining(NJ)method was used to construct a phylogenetic tree with the homologous sequences of E75 from other insects.Specific primers of three LmE75 isoforms were designed using Primer 3.0 software.Reverse transcription-quantitative PCR(RT-qPCR)was used to analyze the expression characteristics of three LmE75 isoforms in different tissues and developmental days from 4th to 5th instar nymphs.To further test whether LmE75 was regulated by 20-hydroxyecdysone(20E),the expression of LmE75 was detected by RT-qPCR after injected with 20E in vivo and interfered with the 20E receptor gene LmEcR by RNAi.The double-stranded RNA(dsRNA)of the common region of three LmE75 isoforms was synthesized in vitro,and then was injected into the 5th instar nymphs.The total RNA of integument was extracted after 48 h,and the first strand cDNA was synthesized as the template of RT-qPCR.The silencing efficiency of LmE75 was detected by RT-qPCR technology and the phenotype was observed.The effect on the structure of cuticle after injected with dsLmE75 was analyzed by H&E staining method.【Result】Three cDNA sequences of LmE75 were obtained based on the transcriptome database of L.migratoria.According to structural characteristics,they were named LmE75A,LmE75B and LmE75C,respectively.The GenBank accession numbers were MN584732,MN

关 键 词：飞蝗 核受体 LmE75 蜕皮激素 RNA干扰 

分 类 号：S433.2[农业科学—农业昆虫与害虫防治]