Rhodopsin-like GPCR参与棉蚜解毒相关P450基因表达的调控机制

Metabolic detoxification plays a pivotal role in the occurrence and development of insecticide resistance. The mechanism involved in cytochrome P450-mediated detoxification has been well studied worldwide. However, the mechanisms, which participate in the regulation of multiply downstream P450 gene expression and detoxification via factors involved in the upstream signaling pathway, still remain unknown. Our previous study indicated that insect Rhodopsin-like GPCR gene could regulate multiple P450 gene expression. Based on the information of genome sequence of the cotton aphid, Aphis gossypii, we will identify all the non-sensory Rhodopsin-like GPCR genes involved in GPCR signaling pathway in Aphis gossypii. Subsequently, we will perform qPCR to select the candidate Rhodopsin-like GPCR genes, which are induced after applying neonicotinoid insecticides to the aphids. With the candidate genes, functional studies will be carried out both in vitro and in vivo. Heterologous expression system of Xenopus oocytes, RNA interference technique and transgenic Drosophila system will be introduced for determining and clarifying the function of the Rhodopsin-like GPCR genes successionally. These studies may elucidate the mechanisms involved in the detoxification and regulation of multiple P450 genes by Rhodopsin-like GPCR, as an upstream factor mediated GPCR signaling pathway. Furthermore, the findings of this research may provide us for a more efficient molecular target, which can manipulate multiple target genes, in turn, control insect pests and insecticide resistance.

昆虫解毒代谢在抗药性的发生和发展中发挥着关键作用，国内外对重要解毒酶系P450介导的解毒代谢机制进行了深入研究，但是，对于上游基因参与解毒作用及其调控下游P450基因表达的作用机制却知之甚少。我们前期研究发现昆虫Rhodopsin-like GPCR基因能够同时调控下游多个P450基因的表达。本申请拟在棉蚜基因组测序基础上，通过qPCR筛选棉蚜中杀虫剂诱导后上调的Rhodopsin-like GPCR基因；利用爪蟾卵母细胞表达系统和RNAi技术及转基因果蝇体系，对Rhodopsin-like GPCR基因的体外和体内功能进行分析，明确棉蚜Rhodopsin-like GPCR基因的功能及其参与下游多个解毒相关P450基因表达的调控机制。为从分子水平筛选和设计“通过控制一个靶标基因来控制多个基因”的高效分子靶标提供理论依据和技术支撑。

棉蚜Aphis gossypii (Glover)属半翅目蚜科，是棉田重要农业害虫。本研究对棉蚜的抗性机理及毒理学特性进行研究。基于棉蚜全基因组和生物信息学分析，对棉蚜GPCR和nAChRs亚基进行鉴定。发现非感知GPCR基因50多个，其中Rhodopsin-like GPCR基因16个。通过分子克隆，获得了8个nAChRs亚基，包括7个α亚基和1个β1亚基，其中α4亚基和α7亚基存在选择性剪切体。克隆得到的所有nAChRs亚基序列具有6个与配体结合有关的Loops（LoopA-F），以及四个与离子通道形成有关的跨膜序列（TM1-4）。同时在α亚基序列中，含有2个典型的α亚基的半胱氨酸的残基。进化树分析表明，棉蚜的各亚基序列与其它昆虫相应亚基具有很高同源性。新烟碱类杀虫剂作为nAChRs的选择性激动剂拥有硝基（-NO2）和氰基(-CN)基团，我们选择代表性的三种新烟碱类杀虫剂对棉蚜nAChRs α1亚基进行结构与功能研究，发现吡虫啉、噻虫嗪和氟啶虫胺氰引发的最大内部电流分别是乙酰胆碱的9.35% ,48.9% 和 81.3%，噻虫嗪和氟啶虫胺氰的EC50与乙酰胆碱的相似，而吡虫啉是其乙酰胆碱的1.76倍，此三种新烟碱类杀虫剂对棉蚜α1亚基的EC50的趋势与生物测定的LC50的趋势是一致的。外源表达系统对呋虫胺的研究中发现，浓度变化引发的内部的电流几乎没有变化，推测棉蚜中呋虫胺的结合位点可能不是α1亚基。手性农药检测棉蚜Agoα1和Agoα8亚基，其中Agoα1-ratβ2对rac-呋虫胺, S-(+)-呋虫胺和 R-(–)- 呋虫胺这三种药剂的刺激没有发生显著性的变化，而Agoα8-ratβ2的检测中，发现其对R-(–)- 呋虫胺单体的剂量浓度曲线相比于单体S-(+)-呋虫胺发生明显的左移，说明R-(–)- 呋虫胺单体对靶标昆虫棉蚜的敏感程度要高于S-(+)- 呋虫胺。