棉铃虫CYP6B6 2-十三烷酮响应转录因子的筛选及其调控机制研究

Helicoverpa armigera is one of the impeotant agricutural pest in the world. The cytochrome P450 monooxygenases have important roles in the metabolism of endogenous and exogenous compounds. CYP6B6 is a cytochrome P450 isolated from cotton bollworm, which not only participates in endogenous metabolism of some substances which have important physiological functions, such as hormones, fatty acid, but also metabolizes some xenobiotics such as allelochemicals and insecticides. Transcription of CYP6B6 can be enhanced by toxic compounds, leading to increased expression of the protein and thus increased detoxi?cation, to make the cotton bollworm possess high tolerance to insecticides.In recent year, the fouctional genes of the detoxic enzyems have been widely investigated, but its regulatory mechanism has not been. On the basis of the previous work we intend to use yeast one-hybrid system to clone the transcription factor that can interacting with the bait sequence, the cis-acting element, which is in 2-tridecanone-responsive region in the promoter of cytochrome P450 CYP6B6 gene from H. armigera. And then confirm the interaction between bait sequence and the transcription factor by the electropjoretic mobility shift，and also validate the transcription activation of the transcription factor gene by the yeast one-hybrid system. To evaluate the function of the transcription factor, we will investigate the CYP6B6 expression profile, tolerance to insecticides and disturbance to the growing development of the cotton bollworm treated with the dsRNA, from the corresponding transcription factor. These results will elucidate the expression mechanism of CYP6B6, from the cotton bollworm treated with 2-tridecanone, regulated by the transcription factor, then dredge the on/off of 2-tridecanone-stress response and finally find main switch of regulation in the whole stress, which will helped us not only to draw out 2-tridecanone-stress signal transduction pathway and its responsive mechanism, but also provide a new technical method for control of Helicoverpa armigera and other pests.

棉铃虫是重要的世界性农业害虫。细胞色素P450是棉铃虫代谢植物次生物质及杀虫药剂等外源化合物的重要酶系，对P450 CYP6B6基因的转录调控机制进行深入研究具有重要的理论和实践意义。近年来，国内外对农业昆虫解毒酶基因功能进行了大量研究，但涉及解毒酶调控机制的研究很少。本项目拟在前期研究的基础上，以CYP6B6 基因启动子中的2-十三烷酮应答核心区域中顺式作用元件为诱饵，利用酵母单杂交系统克隆与之结合的候选转录因子，然后用凝胶阻滞及转录激活等实验筛选转录因子；最后通过RNAi及qRT-PCR技术对得到的转录因子进行功能验证，研究结果可望阐明在2-十三烷酮胁迫下转录因子调控棉铃虫CYP6B6表达的调控机制。以期通过挖掘调控2-十三烷酮胁迫反应的开关，最终找到引领全局的调控总开关，为揭示棉铃虫响应2-十三烷酮胁迫信号转导途径及其应答机制，探索棉铃虫等重要害虫的基因调控抗虫技术手段奠定理论基础。

棉铃虫细胞色素P450 CYP6B6基因在其取食植物时会受到植物次生物质的诱导过表达，增强棉铃虫对外源次生物质和杀虫剂的解毒代谢。在前一个项目的基础上，以2-十三烷酮的响应元件为诱饵，利用单酵母杂交技术和转录组测序(RNA-seq)技术获得诱导CYP6B6表达的候选调控因子，来阐明2-十三烷酮诱导棉铃虫CYP6B6过表达的机制。实验结果表明：基于单酵母杂交技术和转录组测序(RNA-seq)技术获得HaLITAFa、HaLITAFb、HaeIF3G、HaADH5c、HaCAL、HaFKBP12和HaFOXA2作为调控因子与CYP6B6启动子HE1片段结合，同时HaPEBP作为辅因子参与HaeIF3G和HE1片段的结合，这些调控因子和辅因子共同引起CYP6B6的表达发生改变，进而影响棉铃虫对外界刺激的响应及其生长发育。这些结果为CYP6B6调控棉铃虫生长发育提供理论依据，并筛选获得影响棉铃虫生长发育的关键基因，也为建立高效稳定环境友好的害虫控制方法奠定基础。