斜纹夜蛾茚虫威抗性CarEs基因鉴定及其转录调控机制研究

Carboxylesterases (CarEs), as an important family of detoxification enzymes, participate in the metabolism of xenogenous toxicant in almost all agricultural pests and play important roles in the development of insecticide resistance. Current studies showed that increased CarEs activities were important in the development of indoxacarb resistance in Spodoptera litura. However, it is not clear that what are the key important CarEs genes and its regulatory mechanism in the indoxacarb resistance of S. litura. For this question, this study preliminary screening overexpressed CarEs genes in two indoxacarb resistant strains by digital gene expression (DGE) testing, and then clone and sequence analysis to clarify its molecular biological characteristics. Systematically apply qPCR to detect the expression profiles of CarEs genes among developmental stages, different tissues and insecticides treatments. Then, the RNAi and heterogenous expression technology are used to analysis the roles of overexpressed CarEs in indoxacarb resistance of S. litura. The relationship between major xenobiotic transcription factors and resistance CarEs genes and the binding sites of transcription factors are analyzed through RNAi, DNA pull down technology and luciferase reporter assays system. Finally, the results will clarify the molecular mechanism and transcriptional regulation mechanism of CarEs in indoxacarb resistance of S. litura from both gene and protein level.

羧酸酯酶（CarEs）作为一类重要的解毒代谢酶，广泛参与了害虫对外源毒素的代谢过程，在害虫抗性发展中具有重要作用。目前研究表明斜纹夜蛾对杀虫剂茚虫威抗药性的产生与CarEs活性升高有关，但是起关键作用的CarEs基因及其调控机制都还不清楚。针对该问题，本项目拟通过数字基因表达谱（DGE）筛选出在室内茚虫威抗性品系和野外茚虫威抗性品系中共同上调表达的CarEs基因，克隆并序列分析，明确其分子生物学特性；利用qPCR检测这些基因在不同发育阶段、不同组织部位的基本表达特征以及对药剂刺激的应激表达模式；通过RNAi技术和异源表达技术分析过表达的CarEs基因在斜纹夜蛾茚虫威抗性中的作用；再通过RNAi技术、DNA pull down技术和荧光素酶报告系统解析转录因子对抗性CarEs基因的调控关系和调控结合位点。最终从基因和蛋白层面阐明CarEs介导的斜纹夜蛾茚虫威抗药性的分子机制和转录调控机制。

羧酸酯酶（CarEs）作为一类重要的解毒代谢酶，广泛参与了害虫对外源毒素的代谢过程，在害虫抗性发展中具有重要作用。目前研究表明斜纹夜蛾对杀虫剂茚虫威抗药性的产生与羧酸酯酶活性升高有关，但是起关键作用的羧酸酯酶基因及其调控机制都还不清楚。本项目通过生物测定和增效剂实验明确了斜纹夜蛾对茚虫威的抗性水平以及羧酸酯酶在抗性形成中的作用。通过高通量测序和qPCR分析获得了6个在茚虫威抗性品系中上调表达的羧酸酯酶基因，系统发育分析发现6个羧酸酯酶基因广泛分布到3个家族，SlituCXE1、SlituCXE7和SlituCXE12分属到Lepidopteran esterase中，SlituCXE分属到α-esterase中，SlituCXE5和SlituCXE16分属到Integument esterase中。利用RNAi和转基因果蝇技术表明5个羧酸酯酶基因（SlituCXE、SlituCXE1、SlituCXE7、SlituCXE12和SlituCXE16）在斜纹夜蛾对茚虫威抗性形成中发挥了重要作用。进一步克隆获得了斜纹夜蛾转录因子SlituCncC，qPCR检测了SlituCncC在斜纹夜蛾不同品系、发育阶段、组织部位和茚虫威诱导下的表达水平。最后通过RNAi和转录因子结合位点分析表明SlituCXE、SlituCXE1和SlituCXE7的表达受到SlituCncC的调控。研究结果阐明了斜纹夜蛾羧酸酯酶介导茚虫威抗性的分子机制及其调控机制，丰富和发展了害虫抗性机理研究的科学理论体系，实践上可为杀虫剂的田间使用以及抗性治理提供理论依据。