草地贪夜蛾Bt毒素受体基因SfABCC2表达调控研究

草地贪夜蛾入侵我国并不断蔓延，生物防治是控制其危害的重要手段之一。ABCC2转运蛋白是苏云金杆菌（Bt）毒素蛋白的重要受体，申请人已经克隆草地贪夜蛾SfABCC2基因，并解析其作为Bt毒素受体相关功能，但该基因的表达与调控机制尚待研究。本项目拟开展SfABCC2基因转录调控的重要元件-启动子的研究。以SfABCC2启动子区为诱饵利用酵母单杂技术在草地贪夜蛾中肠cDNA文库中筛选调控该基因的转录因子。采用昆虫细胞表达系统和双荧光素酶报告系统确认候选调控蛋白对启动子活性的调控作用。结合生物信息学分析，采用一系列截短的启动子/报告基因载体，鉴定SfABCC2基因启动子核心区域与候选转录因子关键结合位点。然后分别在细胞水平过表达转录因子和活体水平采用RNA干扰技术进行验证。该项目旨在揭示转录因子对SfABCC2基因表达调控作用，为阐明草地贪夜蛾对Bt毒素的抗性机制和科学使用Bt杀虫剂提供理论依据。

Fall armyworm, Spodoptera frugiperda (J. E. Smith) has invaded and spread throughout all major areas in China. Biological control is one of the important means to control its damage. ABCC2 transporter has been shown to be an important receptor protein for Bacillus thuringiensis (Bt) toxin crystals. In our previous study, we cloned the ABCC2 gene, which confirmed that its protein ABCC2 transporter is an important receptor of Bt toxin protein. However, the mechanism of the gene expression and regulation needs to be further studied. This project intends to research the promoter of the crucial element of SfABCC2 gene transcriptional regulation. Firstly, by means of one-hybrid screens, transcription factors expressed from midgut cDNA expression libraries of S. frugiperda can be identified due to the interactions with promoter DNA as nucleic acid bait. Insect cell expression system and dual-luciferase report gene system were used to confirm the regulatory effect of candidate regulatory protein on promoter activity. Subsequently, combined with bioinformatics analysis, a series of promoter and reporter gene vectors were used to identify the core functional regions of the SfABCC2 gene promoter and key binding sites of transcription factors. Finally, overexpression of transcription factors at cellular level and RNAi technology at insect level were used to verify its function, respectively. The purpose of this study was to reveal the role of transcription factors in regulating SfABCC2 gene expression. It may provide a theoretical basis for illuminating the resistance mechanism of Bt toxin and the scientific use of Bt pesticides in S. frugiperda.