细胞素色P450 CYP6CY3基因启动子区变异介导的禾谷缢管蚜对吡虫啉抗性的分子机制

P450基因过量表达介导了很多昆虫的抗药性，但其过量表达的调控机理在农业害虫中鲜有报道。申请者发现，禾谷缢管蚜CYP6CY3的过量表达与吡虫啉抗性密切相关，但不同品系具有不同的抗性发展趋势。CYP6CY3基因启动子区的微卫星多态性及其他变异，是否为造成品系间抗性发展趋势差异的原因，目前尚不明确。本项目拟运用RNAi验证该基因在抗性中的作用；克隆其启动子区，分析抗性品系启动子区变异；构建该基因启动子报告基因载体，确定启动子变异对其活性的影响，明确启动子区变异在不同品系抗性形成中的作用；检测田间种群启动子变异，将田间启动子突变的个体建立单系，并用吡虫啉选择，根据单系抗性及基因表达水平变化，确证启动子变异与抗性的关系；本项目理论上可阐明禾谷缢管蚜对吡虫啉的代谢抗性机制及CYP6CY3的调控进化机理，实践上可以建立基于CYP6CY3启动子变异的该虫抗性预警的分子标记，这对于该虫抗性治理有重要意义。

Though over-expression of the P450 genes regulates high resistance of insects to varies types of insecticides, the molecular mechanism of the over-expression was rarely reported in agricultural pest insects. From the previous work, we found three Rhopalosiphum padi strains developed different resistance to imidacloprid under the same selection environment. The activity of P450 significantly increased in the three resistant strains. The mRNA of CYP6CY3 was over-expressed in resistant strains and was significantly related to the resistance of imidacloprid. In this project, we will use RNAi to silence CYP6CY3, and the susceptibility of the gene silenced strain to imidacloprid will be investigated, clone the promoter region of CYP6CY3, and analyze the cis-acting mutation of promoter in the resistant R. padi strain. The Dual-luciferase reporter assay system will be used to research the effect of mutations in the promoter region on the activity of CYP6CY3. Mutation rate of the promoter region of CYP6CY3 will be investigated in the R. padi field population. To research how the mutation of CYP6CY3 promoter region regulate he resistance development of field population, we will set up clones for those individuals who has mutation, and select the clones with imidacloprid. This study is not only important to understant the mechanism of CYP6CY3-modulated resistance of R. padi to imidacloprid, but also illuminate the regulatory evolution of CYP6CY3, and provide a molecular marker for monitoring the resistance of this pest to the pesticide. This project can obtain sound knowledge for the resistance management of R .padi.