赤拟谷盗章鱼胺受体3（TcOctβR3）cDNA克隆、表达及功能

doi:10.3864/j.issn.0578-1752.2018.07.009

摘要/Abstract

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GenBank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TcOctβR3的cDNA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（TcRPS3）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TcOctβR3，进而利用第二信使cAMP含量测定技术分析TcOctβR3与配体的结合能力。最后，通过体外合成赤拟谷盗TcOctβR3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TcOctβR3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，TcOctβR3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，TcOctβR3在中枢神经系统的表达量显著高于其他组织；赤拟谷盗幼虫在经饥饿处理24 h的过程中，TcOctβR3的表达量呈先下降后上升的趋势，且在处理6 h的表达量最低，为对照的0.47倍，在16 h表达量最高，为对照的1.80倍，最后恢复到正常水平。通过HEK293细胞异源表达TcOctβR3后，cAMP测定结果表明章鱼胺（OA）呈浓度依赖性地激活TcOctβR3，其EC50为8.68×10-7 mol·L-1，萘甲唑啉（NA）的激动活性最高，其有效中浓度EC50为8.56×10-8 mol·L-1。4种供试生物胺激动剂活性强弱为：萘甲唑啉>酪胺（TA）>章鱼胺>多巴胺（DA）。进一步采用RNA干扰技术的分析结果表明，注射dsRNA能有效抑制TcOctβR3的表达，沉默效率高达61.5%，但干扰该基因表达后不会影响赤拟谷盗成虫的爬行速度和产卵量。【结论】TcOctβR3在赤拟谷盗中枢神经系统可能发挥重要作用，能调节幼虫对饥饿胁迫的响应。明确了TcOctβR3的分子生物学特性，可为将来以其为靶标筛选高效激动剂和抑制剂提供理论依据。

关键词: 赤拟谷盗, 章鱼胺受体, 表达模式, 生物活性, 生理功能

Abstract: 【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 GenBank (XP_008198078), the cDNA of an octopamine receptor (TcOctβR3) 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 (TcRPS3) was used as an internal reference. qRT-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. TcOctβR3 was transiently expressed in human embryonic renal cell (HEK293) by using heterologous expression system, and cAMP measuring method was performed to determine the activity of its ability to combine with ligands. Finally, the double stranded RNA of TcOctβR3 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 TcOctβR3 was cloned with open reading frame (ORF) of 1 305 bp, encoding 434 amino acids, with a signature of 7 transmembrane domains which belongs to the superfamily of G-protein coupled receptors. This gene exhibited a close relationship with the AtOctβR3 from Aethina tumida based on neighbor-joining phylogenetic tree. qRT-PCR results indicated that TcOctβR3 was expressed at all tested developmental stages, particularly high at the early larval stage. However, the expression of TcOctβR3 was not significantly different from other developmental stages. Besides, the expression of TcOctβR3 was remarkably higher in the central nervous system (CNS) than that in other tissues. In the process of starvation for 24 h, the expression of TcOctβR3 significantly fluctuated and then returned to the normal level. The lowest expression of TcOctβR3 was 0.47 fold at 6 h and the highest expression was 1.80 folds at 16 h compared with control, respectively. Based on cyclic AMP response assay, it was found that TcOctβR3 could be activated by octopamine (OA) in a dose-dependent manner with a median effective concentration (EC₅₀) of 8.68×10^-7mol·L^-1 after heterogenous expression in HEK293 cells. Naphazoline (NA) strongly activated this receptor with EC₅₀of 8.56×10^-8 mol·L^-1 compared with other ligands. Generally, the rank order for the potency of 4 tested ligands was naphazoline＞tyramine (TA)＞octopamine＞dopamine (DA). RNAi results indicated that the transcript level of TcOctβR3 was significantly knocked down 61.5% by injected the targeted dsRNA. However, no effect on the walking speed and fecundity of T. castaneum adults was observed. 【Conclusion】TcOctβR3 plays an important role in the central nervous system, which modulates the response of the beetle larvae upon starvation. The molecular characterizations were determined based on a cAMP assay, which will provide a solid basis for the future screening of its high efficiency agonists and inhibitors.

Key words: Tribolium castaneum, octopamine receptor, expression profile, biological activity, physiological function

刘小强，蒋红波，李慧敏，熊英，王进军. 赤拟谷盗章鱼胺受体3（TcOctβR3）cDNA克隆、表达及功能[J]. 中国农业科学, 2018, 51(7): 1315-1324.

LIU XiaoQiang, JIANG HongBo, LI HuiMin, XIONG Ying, WANG JinJun. The cDNA Cloning, Expression Profiling and Functional Characterization of Octopamine Receptor 3 (TcOctβR3) in Tribolium castaneum[J]. Scientia Agricultura Sinica, 2018, 51(7): 1315-1324.

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