桃蚜对吡虫啉抗性相关基因GroEL的克隆与功能研究

害虫产生抗药性是其对杀虫剂适应的一种普遍策略，明确抗药性的分子机制是解决害虫抗药性问题的关键。在前期研究中，项目申请人采用蛋白质组学方法，从桃蚜的吡虫啉敏感和抗性品系中分离、鉴定了新的抗性相关蛋白GroEL。迄今尚未见该蛋白与抗药性相关的功能报道。在上述研究结果基础上，本项目通过克隆桃蚜GroEL基因，比较敏感和吡虫啉抗性品系GroEL基因在核苷酸及氨基酸序列上的差异；检测桃蚜敏感和抗性品系GroEL基因的mRNA表达量，明确抗性形成与GroEL基因表达的关系；构建GroEL基因的原核表达载体，体外表达桃蚜GroEL基因；利用RNAi技术研究GroEL基因低表达情况下桃蚜敏感品系对吡虫啉的毒力，验证GroEL基因在吡虫啉抗性形成中的作用。以上研究结果不仅能够揭示桃蚜对吡虫啉新的抗性分子机制，而且为桃蚜对吡虫啉抗性的早期诊断、抗性治理以及GroEL基因的合理利用提供理论依据。

The development of resistance in pests is a universal adaptation strategy responding to insecticides. Elucidating the molecular mechanisms of insecticide resistance is the key to solve the problem of insecticide resistance. In our previous study, a new protein GroEL that may be associated with resistance was isolated and identified from imidacloprid-susceptible and resistant strains of Myzus persicae by proteomics. However, to our knowledge there is no published report regarding the relation between GroEL and insecticide resistance. Based on the results of the above studies, the missons of the project are listed as follows: （i）cloning the full length cDNA of GroEL gene from M. persicae, and finding the differences in nucleotide and amino acid sequence of GroEL gene in imidacloprid-susceptible and resistant strains. （ii）analysing the mRNA expression of GroEL gene in susceptible and resistant strain using the real-time quantitative PCR, to clarify the relationship between the development of resistance and the expression of GroEL gene. （iii）constructing GroEL gene into prodaryotic expression vectors pET28a(+) so as to be expressed in vitro. （iv）investigating the effects of RNAi-mediated gene silening on the insecticidal toxicity of susceptible aphids, and verifying the role of GroEL gene on imidacloprid resistance. The result of the experiment will help us to understand a new molecular mechanisms of imidacloprid resistance in M. persicae. It will also provide a theoretical basis for early detection and management of imidacloprid resistance, and for rational utilization of GroEL gene.