赤拟谷盗章鱼胺受体3(TcOctβR3)cDNA克隆、表达及功能  

The cDNA Cloning,Expression Profiling and Functional Characterization of Octopamine Receptor 3(TcOctβR3) in Tribolium castaneum

作　　者：刘小强 蒋红波[1] 李慧敏 熊英 王进军[1] LIU XiaoQiang;JIANG HongBo;LI HuiMin;XIONG Ying;WANG JinJun(Key Laboratory of Entomology and Pest Control Engineering of Chongqing, College of Plant Protection, Southwest University, Chongqing 400716)

机构地区：[1]西南大学植物保护学院昆虫学及害虫控制工程重庆市市级重点实验室,重庆400716

出　　处：《中国农业科学》2018年第7期1315-1324,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金面上项目(31772233)

摘　　要：【目的】章鱼胺信号系统在调节昆虫行为和生理过程中具有至关重要的作用。赤拟谷盗(Tribolium castaneum)作为一种模式昆虫,被广泛用于解析昆虫生长发育及生理等调控机制的研究工作。本研究以赤拟谷盗为对象,旨在明确章鱼胺受体在调节赤拟谷盗行为和生理方面的功能。【方法】根据Gen Bank登录的相关序列信息(XP_008198078),利用RT-PCR技术克隆赤拟谷盗章鱼胺受体基因Tc OctβR3的c DNA序列。利用在线生物信息学分析软件预测该基因的开放阅读框、编码的氨基酸序列以及跨膜结构域等信息,基于邻接法构建该基因与其他昆虫相关序列的系统发育树,明确系统进化关系。分别提取赤拟谷盗各发育阶段(卵、幼虫、蛹和成虫)、不同组织(中枢神经系统、脂肪体、中肠、后肠、马氏管、精巢和卵巢)以及饥饿胁迫后的RNA,以赤拟谷盗核糖体蛋白S3(Tc RPS3)为内参基因,采用实时定量PCR技术分析该基因在赤拟谷盗不同发育阶段、不同组织以及在饥饿胁迫下的表达模式。运用哺乳动物异源表达系统在人胚胎肾细胞HEK293中瞬时表达Tc OctβR3,进而利用第二信使c AMP含量测定技术分析Tc OctβR3与配体的结合能力。最后,通过体外合成赤拟谷盗Tc OctβR3的双链RNA,利用RNA干扰以及轨迹球行为分析等技术探究该基因的生理功能。【结果】序列分析结果表明,赤拟谷盗Tc OctβR3开放阅读框全长1 305 bp,编码434个氨基酸,序列中含有G蛋白偶联受体典型的7个跨膜结构域。基于邻接法构建的系统发育树表明,该基因编码的蛋白质与小蜂甲(Aethina tumida)的OctβR3亲缘关系最近。实时定量PCR分析结果表明,Tc OctβR3在赤拟谷盗各发育阶段均有表达,尤其在低龄幼虫期转录水平最高,而在其他发育阶段表达量无显著差异;在赤拟谷盗不同组织中,Tc OctβR3在中枢神经系统的表达量显著高于其他组织;赤拟谷盗幼虫【Objective】Octopaminergic signaling system plays a crucial role in the reregulation of behavioral and physiological processes in insects. The red flour beetle（Tribolium castaneum） is a model insect which has been widely used in the study of insect growth, development and physiology. The objective of this study is to utilize T. castaneum as the research insect and elucidate the vital functions of octopamine receptors involved in the physiology and behavior.【Method】Based on the sequence information in Gen Bank（XP008198078）, the c DNA of an octopamine receptor（Tc OctβR3） was cloned by RT-PCR. The open reading frame（ORF）, deduced amino acid sequence and the membrane structure domains were predicted by using online service, and phylogenetic tree associated with OctβR3 from other insects was constructed by using neighbor-joining method to clarity its phylogenetic relationship. In addition, the RNA was extracted from different developmental stages（egg, larva, pupa, and adult）, different tissues（central nervous system, fat body, midgut, hindgut, malpighian tubule, testis and ovary）, as well as the larvae under stress of starvation, respectively. Ribosomal protein S3 gene（Tc RPS3） was used as an internal reference. q RT-PCR（real-time quantitative PCR） was employed to determine its expression patterns in different developmental stages, different tissues as well as the induced expression profiles under the stress of starvation. Tc OctβR3 was transiently expressed in human embryonic renal cell（HEK293） by using heterologous expression system, and c AMP measuring method was performed to determine the activity of its ability to combine with ligands. Finally, the double stranded RNA of Tc OctβR3 was synthesized in vitro, and the physiological functions were verified by track ball behavior analysis as well as RNA interference（RNAi） technology. 【Result】 A complete sequence of Tc OctβR3 was cloned with open reading frame（ORF） of 1 305 bp, encoding 434 amino acids, with a

关 键 词：赤拟谷盗 章鱼胺受体 表达模式 生物活性 生理功能 

分 类 号：S379.5[农业科学—农产品加工]