华北大黑鰓金龟对6种气味分子的识别机理

Holotrichia oblita Faldermann is an important agricultural pest which has caused serious economic damage in China. Its larvae were controlled difficultly and host plant volatiles are going to be a good method to controlling adult. The goal of this project is to elucidate the molecular recognition mechanism based on OBPs in adult’s antennae. The antennal specific OBPs gene will be identified by bioinformatics analysis from antennae transcriptome and quantitative real-time PCR to different tissues. H. oblita showed marked electrophysiological and behavioral responses to 6 odors, cis-3-Hexen-1-ol, trans-2-Hexenol, linalool, Methyl anthranilate, cinnamaldehyde, phenethyl alcohol. Homology modeling and molecular docking combining with up-regulated OBP genes will help us to screen the OBPs which bind well with these 6 odors. OBPs are cloned, expressed, purification, then different factors fluorescence binding assay with a pH-dependent binding assay were applied to identify the functions of OBPs. By using polyclonal antibody and electron microscope, we observed the expression of OBPs in the sensilla of antenna. We also applied fluorescence in Situ hybridization (WM-FISH) to investigate the co-expression of H. oblita OBPs in the sensilla. The binding model between OBP and odor will be cleared and help us to clarify the function of OBPs. Co-immunoprecipitation method will confirm the cooperative interactions between OBPs. Mutants will be gotten by using site-directed mutagenesis in correct site and changes of binding affinity will be tested based on fluorescence assays. Results to date confirm the role of cooperative OBPs in odor perception and discrimination. The function of the candidate OBPs gene will be checked by RNAi technique. Through injection with synthesized dsRNA of target OBPs to pupae, gene expression will be checked by RT-qPCR, and the effect of RNAi will be checked by olfactory behavior of beetles using Y type tube. The results will clarify the molecular recognition mechanism between H. oblita and host plants based on olfactory system, and it is also very important in the development of new strategies to adults control techniques.

华北大黑鳃金龟是我国发生严重的一类地下害虫，幼虫防治困难，利用信息素防治成虫前景广阔。本研究以阐明其成虫触角中OBPs对气味化合物的识别机理为目标，通过分析触角转录组测序数据和OBPs组织表达谱，获得嗅觉相关的OBPs基因；利用同源建模和分子对接以及检测上调OBPs，系统筛选参与识别已知6种活性气味分子的OBPs；原核表达和蛋白纯化，在不同pH下进行单、多因子组合的荧光竞争结合试验，结合免疫电镜和荧光原位杂交技术，明确OBPs与气味分子的结合模式和功能；免疫共沉淀验证OBPs间的互作关系；定点突变互作OBPs与气味结合的位点，检验突变体对气味分子的识别能力，明确OBPs互作对气味分子的识别机制及意义；采用RNAi技术，检测成虫选择行为变化及靶标基因抑制效果，验证OBPs在其体内的功能。研究结果不仅揭示华北大黑鳃金龟对寄主植物嗅觉识别的分子机理，对于开发其成虫行为调控措施也具有重要实践意义。

华北大黑鳃金龟是我国发生严重的一类地下害虫，幼虫防治困难，利用信息素防治成虫前景广阔。本研究通过转录组测序、OBPs原核表达纯化和荧光竞争结合试验，结合免疫电镜和荧光原位杂交技术，明确了OBPs与活性气味分子的结合模式和功能，气味结合蛋白形成二聚体能够增强对化合物的识别能力，氢键在识别中发挥了重要作用；采用RNAi技术，检测成虫选择行为变化及靶标基因抑制效果，验证OBPs在其体内的功能。研究结果表明，华北大黑鳃金龟对植物源化合物的识别主要由HoblOBP1、2、4、9、13来完成。随后，克隆华北大黑鳃金龟触角特异表达的4个HoblORs，分别为HoblOR1、5、6、15，利用爪蟾卵母细胞表达后用双电极电压钳检测ORs对植物源化合物的结合特性，研究结果表明，HoblOR5和HoblOR15均可结合植物源化合物反-2-己烯醇，其中HoblOR15的结合能力更强，利用RNAi技术，验证了华北大黑鳃金龟HoblOR15在气味识别中的功能。研究结果阐明了华北大黑鳃金龟对于6种主要植物挥发物的嗅觉识别机制，也为筛选和研制更加高效的食诱剂和干扰剂奠定基础，还将为开发以为调控行为主的生态防控措施提供新的作用靶标。