二化螟GABA受体的组成及其药理学特性研究

GABA receptor is an important insecticide target but without deep study in the agricultural insect. In recent years, the new compounds (fluralaner and BPB etc.) target to the GABA receptor as well as the conventional insecticides (fipronil, avermectin, etc.) but without cross-resistance were synthesized, however, the binding sites are not clear. To explain the phenomenon, our project aims to: First, clone the GABA receptor subunits, RDL, GRD and LCCH3, from the rice stem borer, compare the difference of three subunits to those from mammalian and detect spatial and temporal distribution expression profile of three subunits; Second, utilize the intracellular loop region (R1, G1 and L1) of subunits as antigen to obtain polyclonal antibodies, and examine the subunits composition of GABA receptor by co-immunoprecipitation method; Third, examine the electrophysiological responses of Xenopus oocytes with GABA receptor to various insecticides using two-electrode voltage clamp technique to identify the GABA receptor pharmacological properties. The completion of this present project will define the natural composition and pharmacological properties of GABA receptors in the rice stem borer and reveal its molecular structure and mechanism of insecticides action for finding the target site from different subunits and providing theoretical basis and technical support for the development and application of new insecticides, which function on the GABA receptor.

针对GABA受体作为农药的重要靶标却在农业昆虫中研究浅显的现状，围绕近几年以GABA受体为靶标的新化合物（fluralaner、BPB等）与传统农药（氟虫腈、阿维菌素等）无交互抗性和作用靶位点不明确这一科学问题。本研究拟：1.选用水稻害虫二化螟为研究对象，克隆其GABA受体RDL、GRD和LCCH3亚基，比对其与哺乳动物亚基的差异，检测其表达谱，揭示受体的时空分布规律；2.采用亚基的胞内环区域片段（R1、G1和L1）为抗原制备多克隆抗体，通过免疫共沉淀技术明确受体的亚基组成；3.利用双电极电压钳技术测定表达GABA受体的爪蟾卵母细胞对多种杀虫剂的电生理反应，明确GABA受体的药理学特性。预期结果可明确二化螟GABA受体的天然组成及其药理学特性，揭示其分子结构和与农药的作用机制，为不同亚基上靶位点的寻找及以GABA受体为靶标的新农药研发和应用提供理论依据和技术支持。

本研究项目通过分子生物学技术和电生理技术研究了二化螟GABA受体的组成及其药理学特性，为不同亚基上靶位点的寻找及以GABA受体为靶标的新农药研发和应用提供理论依据和技术支持。.项目研究内容包括：①克隆二化螟GABA受体RDL、GRD和LCCH3亚基及其同源基因；②明确二化螟GABA受体的组成及其与脊椎动物中GABAA受体亚基的差异；③弄清二化螟GABA受体亚基与杀虫剂作用中的药理学特性；④总结分析。.通过该项目，申请人利用分子克隆技术成功获得了二化螟GABA受体的RDL、8916和LCCH3亚基；利用荧光定量PCR明确了多个不同亚基的时空表达谱，并利用免疫共沉淀技术探究了亚基的分布和结合模式；利用爪蟾卵母细胞体外表达，确定了RDL不同亚型以及8916和LCCH3亚基的功能与特点，明确了这些体外通道与杀虫剂（传统和新型）的结合情况。项目证实，鳞翅目昆虫中不含有GRD基因，却含有8916基因；而RDL1和RDL2亚基可以共存于一个体表明RDL亚基的关键结合位点，A-S之间氨基酸突变是自然存在的，而杀虫剂可能只是起到了筛选的作用；GABA受体的各个亚基mRNA的相对表达量随着龄期的增长呈下降趋势，这与其他昆虫内GABA受体mRNA表达模式相似；而爪蟾卵母细胞的异源表达的结果表明，8916基因和LCCH3基因可以形成功能性离子通道，这也是首次报道的，除RDL亚基以外的GABA受体亚基可以形成功能性阴离子受体通道；传统杀虫剂与新开发的杀虫剂与体外靶标受体结合的差异说明，两者之间作用方式存在差异，无交互抗性风险。以上结果为我们更深层次地认识昆虫GABA受体的普遍性和特异性提供了重要的数据。截至该报告提交，已发表相关SCI论文8篇，申请专利2项，培养研究生3人。