赤拟谷盗Pyrokinin受体的鉴定及其生理功能研究

The G protein-coupled receptors (GPCRs) constitute a large and ancient superfamily of integral cell membrane proteins that play a central role in signal transduction. They are responsible for the communication between insect cells and the environment and regulate the behavior, growth, development, reproduction and immunity in insects. Due to their important physiological functions in insects and their potential as novel insecticides targets, insect GPCRs have been paid more and more attention, which made it an international hot topic. Insects PK/PBAN receptors are typical GPCRs, it has been reported that pyrokinin receptors could regulate the growth and development in Drosophila, while PBAN receptors regulated the reproduction behaviors via controlling the production of female sex pheromone in moths. Based on the genome sequence of Tribolium castaneum, 3 pyrokinin-like receptors were predicted, however, no any information about their functions in beetles is available so far. In the current research, advanced molecular techniques such as RNAi, qPCR and heterologous expression will be employed to reveal the physiological functions of the 3 pyrokinin-like receptors in Tribolium castaneum. The results will increase our knowledge about insect GPCRs, facilitate our understanding of the development, reproduction and their regulation mechanisms in beetles. Furthermore, high-throughput screening system of the agonists and antagonists for Tribolium pyrokinin receptors could be established, which will provide basic information for the development of novel insecticides targeted on insect GPCRs.

昆虫GPCR负责细胞与环境、细胞之间的通讯，调节昆虫的行为、生长、发育、生殖以及免疫反应等。为探明GPCR在昆虫体内的重要生理功能，深入发掘昆虫GPCR作为新型杀虫剂作用靶标的潜力，该领域的研究越来越受到研究人员的重视。昆虫PK/PBAN受体是典型的GPCR，研究表明其调控果蝇的生长、发育，调控蛾类性外激素的合成及生殖行为。赤拟谷盗基因组中预测有3个pyrokinin-like receptor，然而其功能却一无所知。本研究拟采用RNAi、qPCR以及CHO细胞表达等手段，鉴定赤拟谷盗pyrokinin receptor，揭示其在赤拟谷盗体内的重要生理功能。研究结果将增强对昆虫GPCR功能的认识，促进理解赤拟谷盗及其他甲虫种类发育、繁殖的调控机制；并据此建立基于赤拟谷盗Pyrokinin receptor抑制剂和激发剂高通量筛选平台，为以昆虫GPCR为靶标的新型杀虫剂创制提供理论依据。

在本项目的资助下，我们以模式昆虫赤拟谷盗为研究对象开展了PRXamide神经肽（CAPA、PK1/DH、PK2/PBAN和ETH）及其受体（分别是CAPAr、PKr-A、PKr-B、PKr-C以及ETHr）的分子生物学、药理学特性以及功能表达等进行了深入的研究。研究内容主要包括：一是基于赤拟谷盗基因组数据，采用RT-PCR技术克隆了赤拟谷盗PRXamide神经肽及其受体cDNA，并对其序列进行了分析；利用实时定量PCR技术解析了PRXamide受体的时空表达模式。二是在中国仓鼠卵母细胞中成功表达了赤拟谷盗PRXamide受体，基于细胞内钙离子流动测定的方法验证了其功能、鉴定了模式配体并确定了其配体范围。三是与美国农业部Ronald Nachman博士合作，基于PRXamide受体活性测定的方法检测了人工合成的30余个多肽模拟物对赤拟谷盗PRXamdie受体的激动和拮抗活性。四是通过比较赤拟谷盗PRXamide受体功能测定与系统发育分析数据，发现了赤拟谷盗PRXamide——受体间协同进化过程中的多样性。研究结果增加了我们对昆虫PK/PBAN receptor的认识，促进理解赤拟谷盗及其他甲虫种类发育、繁殖的调控机制；为基于昆虫GPCR为靶标的新型农药创制提供理论依据，并有可能获得抵御鞘翅目农业害虫的更好方法。. 除圆满完成了原定研究计划外，还在赤拟谷盗中证实了Orcokinin两种剪切子（OKA和OKB）的存在，利用原位杂交、免疫组化等手段探究其在神经系统内的分布，应用RNAi技术分别沉默OKA、OKB或者将二者同时沉默后，赤拟谷盗成虫需要更长时间从假死状态中“觉醒”；幼虫则需要更长时间从溺水昏迷状态中恢复。这说明OKA和OKB极有可能参与调节赤拟谷盗的应激行为，研究结果为揭示赤拟谷盗适应环境胁迫的分子生理学机制提供参考。