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

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.

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。

禾谷缢管蚜是我国麦类作物的重要害虫，杀虫剂广泛使用导致的抗药性是其危害逐年加重的重要因素。P450基因的过量表达是害虫对吡虫啉等农药产生抗药性的重要原因。本研究监测了我国不同地区禾谷缢管蚜田间种群对吡虫啉等农药的抗性水平；建立了禾谷缢管蚜室内吡虫啉抗性品系；分析了禾谷缢管蚜对吡虫啉的抗性机制，发现CYP6CY3 等P450基因表达水平和活性上升是禾谷缢管蚜对吡虫啉产生抗性的重要原因；通过基因组和转录组数据分析，并结合RACE技术，获得了禾谷缢管蚜55个P450基因的基因序列；克隆获得3个禾谷缢管蚜CYP6CY3基因全序列，并分析了CYP6CY3基因在不同抗性品系中的表达模式；运用基因步移技术克隆了CYP6CY3-3基因启动子区，分析禾谷缢管蚜抗性和敏感单克隆CYP6CY3-3基因启动子区多态性及变异，同时构建表达载体，分析了CYP6CY3-3基因启动子活性，以及启动子变异与抗性的关系；研究了禾谷缢管蚜P450还原酶RpCPR基因的突变，发现RpCPR基因突变与禾谷缢管蚜对吡虫啉的抗性具有重要关系；分析了P450基因介导的禾谷缢管蚜对其他农药的抗性机制，发现RNA干扰CYP4CJ1基因可以显著影响禾谷缢管蚜对异丙威的敏感性。本研究结果对于深入阐明禾谷缢管蚜对吡虫啉等农药的代谢抗性机制及该虫的抗性治理具有重要的意义。