小菜蛾PxPPO基因的表达及转录调控机理研究

Phenoloxidase (PO) has important physiological functions in the process of growth and development of insects. This project will carry out the study of the diamondback moth PxPPO gene expression and transcriptional regulation mechanism. This study intends to build PxPPO prokaryotic, eukaryotic expression vector, to clone the promoter of PxPPO, and to determine the structure character (including M-box, E-box, etc.) of the promoter. And, the study tries to clone the Microphthalmia-associated Transcription Factor (MITF) and to carry out its functional verification. This project by applying Chromatin Immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) to determine the specific binding relationship of MITF with the cis-acting elements, and to analyze the molecular mechanism of MITF up-regulating the phenoloxidase activity. The launching of the project is to lay the foundation for the phenoloxidase gene engineered, and to provide new ideas for the genetically engineered pesticides.

昆虫酚氧化酶（PO）在昆虫的生长发育过程中具有重要的生理功能。本项目拟在前期研究的基础上，开展小菜蛾PxPPO基因的表达及转录调控机理研究。本研究拟构建PxPPO的原核、真核表达载体；克隆PxPPO1和PxPPO2的启动子，明确启动子的结构组成（M-box、E-box等）；克隆小眼畸形相关转录因子（MITF）并进行检测；应用染色质免疫沉淀技术（ChIP）和电泳迁移率变动实验（EMSA）研究小眼畸形相关转录因子（MITF）与启动子结构元件特异性结合的关系，从而探讨小眼畸形相关转录因子（MITF）参与的上调酚氧化酶活性的分子机理。本项目的开展可为酚氧化酶基因工程改造等研究奠定基础，为基因工程农药提供新思路。

昆虫酚氧化酶（PO）在昆虫的生长发育过程中具有重要的生理功能，开展小菜蛾PxPPO基因的表达及转录调控具有重要的科学意义。本项目开展了PxPPO基因的体外表达研究，获得了PxPPO1和PxPPO2离体重组蛋白，分子量分别为83 kDa（PxPPO1）和84.5 kDa（PxPPO2），经检测具有酶活性；明确了PxPPO1和PxPPO2在小菜蛾不同虫态、虫龄的体内表达规律，显示PxPPO1和PxPPO2在卵和4龄幼虫中表达量最高，在成虫中表达量最低；明确了槲皮素、曲酸和球孢白僵菌等对PxPPO1和PxPPO2基因转录表达活性的影响；探讨了PPO的转录表达水平与类绛色细胞的密度之间存在的相关性；通过生物信息学分析，明确了PxPPO1和PxPPO2的启动子区域及结构组成；克隆了小眼畸形相关转录因子（MITF）cDNA序列。本项目初步探明了PxPPO基因的表达规律和转录调控机制，为深入探讨MITF与启动子结构元件的特异性结合奠定了基础。