梨小食心虫生物钟基因Gmper和Gmtim的鉴定及其对羽化节律的影响

Identification of circadian clock genes Gmper and Gmtim and their contributions to the emergence rhythm in Grapholita molesta(Lepidoptera:Tortricidae)

【目的】本研究旨在探究梨小食心虫Grapholita molesta生物钟基因Gmper和Gmtim的分子特性与表达模式,分析其对羽化节律的调控作用,为梨小食心虫的防控提供潜在新靶标。【方法】根据梨小食心虫转录组数据,采用PCR技术克隆生物钟基因Gmper和Gmtim的cDNA全长;利用RT-qPCR测定这两个基因在梨小食心虫成虫头、胸、腹和足中的表达量,及其在蛹头部中的日表达模式;应用siRNA进行RNAi技术分析Gmper和Gmtim在梨小食心虫羽化节律中的作用。【结果】克隆获得梨小食心虫Gmper基因(GenBank登录号:MN862636)和Gmtim基因(GenBank登录号:MN862637)全长cDNA。Gmper基因开放阅读框长2862 bp,编码953个氨基酸,序列中含2个PAS结构域和1个PAC结构域;Gmtim开放阅读框长3048 bp,编码1015个氨基酸。Gmper和Gmtim在雌雄成虫头部中的表达水平均高于其他组织中的;蛹期头部两基因在暗期的表达水平显著高于光期的。RNAi下调这两个基因的表达均导致梨小食心虫羽化时间更加分散以及羽化高峰期的羽化成虫数量显著降低。【结论】梨小食心虫生物钟基因Gmper和Gmtim具有组织和昼夜表达差异性,两基因对梨小食心虫羽化节律的调控有重要作用。研究结果为开发基于发育行为节律调控的梨小食心虫监测和防控方法提供了新线索。

【Aim】The purpose of this study is to investigate the molecular characteristics and expression patterns of two circadian clock genes Gmper and Gmtim in the oriental fruit moth,Grapholita molesta,and to explore their regulatory roles in the emergence rhythm,so as to provide potential new targets for controlling G.molesta.【Methods】Based on the transcriptome data of G.molesta,the full-length cDNAs of two circadian clock genes Gmper and Gmtim were cloned by PCR.The expression levels of these two genes in the head,thorax,abdomen and leg of adults and their daily expression patterns in the pupal head were determined by RT-qPCR.Then the regulatory roles of Gmper and Gmtim in the emergence rhythm of G.molesta were explored by RNAi with siRNAs.【Results】The full-length cDNAs of Gmper(GenBank accession no.:MN862636)and Gmtim(GenBank accession no.:MN862637)were cloned from G.molesta.The open reading frame of Gmper is 2862 bp in length,encoding 953 amino acids with two PAS domains and a PAC domain.The open reading frame of Gmtim is 3048 bp in length,encoding 1015 amino acids.The expression levels of Gmper and Gmtim in heads were higher than those in other tissues of both male and female adults.In pupal head,the expression levels of the two genes were significantly higher in the scotophase than in the photophase.The emergence rhythm changed after RNAi of both Gmper and Gmtim,and G.molesta in the RNAi group showed a more dispersed emergence pattern than that in the control group,and the amount of emerged adults during the emergence peak period decreased after RNAi.【Conclusion】Both Gmper and Gmtim in G.molesta show differential expression patterns among different tissues and between day and night,and they may play important regulatory roles in the emergence rhythm of G.molesta.The results provide clues to the development of monitoring and control technologies for G.molesta based on behavioral rhythm regulation.