Ca2+信号通路中PxRyR和PxCaM的互作及其导致小菜蛾对氯虫苯甲酰胺高抗性产生的机制研究

At present, it is difficult to explain the high resistance of Plutella xylostella to chlorantraniliprole by metabolism and target mutations. As accessory proteins of PxRyR, the down-regulated of PxCaM could affect insecticide susceptibility by changing the conformation of PxRyR. However, whether the interactions between PxRyR and PxCaM could result in high resistance to Chlorantraniliprole or not is still unknown. In order to explore the resistance mechanisms，based on the susceptible strain transcriptome, resistance strains digital gene expressions sequencing analysis of p. xylostella and the information of genes in Ca2+ signaling pathway obtained from the previous work, this project would be conducted to obtain the PxCaM gene expressed efficiently in E. coli and purify the PxCaM-GST and PxRyR proteins，validate the protein-protein interactions between protein PxCaM and PxRyR using GST-Pulldown, define the protein contents of PxCaM which would affect the conformation of PxRyR and silence the expression of PxCaM and PxRyR gene by RNAi silencing. Meanwhile, the sensitivity of P. xylostella and PxRyR to chlorantraniliprole would be tested and the function of PxCaM and PxRyR in the resistance of P. xylostella to chlorantraniliprole would be investigated in our work to clarify the influence and mechanism of the protein to protein in Ca2+ signaling pathway to the resistence of chlorantraniliprole in P. xylostella. The results of the project will provide theoretical basis and evidence for explaining the resistance mechanism of p. xylostella to chlorantraniliprole, enrich and promote the research of pesticides target toxicology and the study of pest resistance mechanism.

小菜蛾钙调蛋白（PxCaM）作为Ca2+信号通路中的附属蛋白，其量变可以改变鱼尼丁受体（PxRyR）构象，并影响杀虫剂的活性，但二者之间的互作是否导致小菜蛾对双酰胺类杀虫剂高抗性产生，尚不明确。为阐明此机理，本项目在已测定小菜蛾转录组和表达谱，获得PxRyR和PxCaM基因信息的基础上，体外高效表达并纯化PxCaM-GST和PxRyR蛋白，GST-Pulldown验证两蛋白之间的互作，明确PxCaM对PxRyR构象影响的蛋白量；RNAi沉默PxCaM和PxRyR基因的表达，并测定小菜蛾和PxRyR对氯虫苯甲酰胺（Chlo）的敏感性，明确PxCaM和PxRyR在小菜蛾对Chlo抗性产生中的功能；阐明Ca2+信号通路中关键基因/蛋白的互作量变对小菜蛾抗药性的影响及机制。项目的结果将为阐述小菜蛾对Chlo的高抗药性机制提供理论基础和证据，丰富并推动杀虫剂靶标毒理与害虫抗药性机理研究。

氯虫苯甲酰胺是一种高效、低毒、环保的杀虫剂，是防控小菜蛾等鳞翅目害虫的理想药剂。但是，小菜蛾在2-3年的时间内就对其产生了高抗药性，使其失去了应有的防控效果。昆虫抗药性机理的研究是抗性治理措施制定的基础，本研究系统探索了小菜蛾鱼尼丁受体蛋白RyR、钙调蛋白CaM、FK系列蛋白和P450代谢解毒酶基因的差异表达和抗药性功能，优化了小菜蛾肌质网膜蛋白的提取技术。研究结果阐明了小菜蛾钙离子信号通路中鱼尼丁受体蛋白、钙调蛋白、FK蛋白和P450代谢解毒酶基因的表达与抗药性的关系。与敏感小菜蛾种群相比，氯虫苯甲酰胺高抗药性小菜蛾种群的鱼尼丁受体蛋白、钙调蛋白和FK蛋白基因的表达量均显著下调，鱼尼丁受体蛋白基因和钙调蛋白基因的表达水平在一定程度上影响了小菜蛾对氯虫苯甲酰胺的抗药性水平。钙调蛋白基因的表达量与抗药性水平成正相关，基因表达量越低，小菜蛾对氯虫苯甲酰胺的敏感性越高；鱼尼丁受体蛋白基因的表达量越低，小菜蛾对氯虫苯甲酰胺的敏感度降低；抗性小菜蛾种群中FK4蛋白基因的表达量显著降低，但FK8蛋白基因的表达量显著提高，FK506蛋白基因的表达水平在抗性和敏感小菜蛾种群中没有差异。应用蔗糖梯度高速离心法和膜蛋白提取试剂盒提取了小菜蛾膜蛋白，并应用2-DE技术对小菜蛾对氯虫苯甲酰胺的抗药性基因进行了研究。鱼尼丁受体蛋白RyR、钙调蛋白CaM基因下调表达是高抗药性小菜蛾种群的一个特征，该特征可用于田间小菜蛾氯虫苯甲酰胺抗性种群的分子监测依据。该项目研究结果丰富了小菜蛾氯虫苯甲酰胺抗药性机理的研究，为小菜蛾抗药性治理措施研究和抗性种群的分子监测提供了理论基础和依据。

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