致倦库蚊嗅觉系统感受不同宿主气味的分子机制研究

As opportunistic blood sucker, Culex pipiens quinquefasciatus, human blood, bird blood and other mammals′blood features complicated blood-sucking habit. Owing to this, Cx. p. quinquefasciatus becomes the important transmitting vector for various pathogens around the world. Recently, how to use the physiological behavior characteristic of Cx. p. quinquefasciatus to develop men prevention and treatment methods has been gradually been focused little by little. In our previous study, we got 12 olfactory receptor (OR) genes of Cx. p. quinquefasciatus possibly responsible for blood sucking habitate by applying high throughput transcriptome sequencing technique and biological information analysis. Among the 12 OR genes, mechanism of 11 genes is stiil unknown. In this stdudy, the role of 11 OR genes in positioning host and blood sucking process of Cx. p. quinquefasciatus is discussed through expression quantitative analysis, electrophysiological experiment and odor compound in vitro combination experiment so as to pave foundation for further clarifying the inner molecular mechanism of blood sucking habit of Cx. p. quinquefasciatus. In the meantime, the molecular targets identifying and combining different host odor compounds and avoiding odor compounds in the olfactory system of Cx. p. quinquefasciatus will be defined and the methods for screening out the odor compounds based on OR protein will be established, which have significant theoretical meaning and application value to construct the molecular model for insect repellents and attractant screening of Cx. p. quinquefasciatus and design new prevention and treatment methods to interfere the association of mosquito and host so that the popularity of mosquito-borne disease.

致倦库蚊既嗜吸鸟血也吸人血，因此在全球成为了多种病原体的重要传播媒介。本课题主要针对致倦库蚊复杂嗜血习性的分子机制进行研究。在我们的前期工作中，应用转录组测序技术，结合生物信息学分析，筛选出了致倦库蚊12个可能与其吸血行为相关的OR基因，其中11个功能未知。本课题拟在前期工作的基础上，针对这11个OR基因，通过蚊虫不同发育时期的基因表达谱分析、体外蛋白表达及其与不同宿主气味化合物（人气味、鸟气味）的体外结合实验，明确致倦库蚊嗅觉系统中识别和结合不同宿主气味物质的分子靶标，探讨这些OR基因在致倦库蚊寻找和定位宿主过程中的作用，为进一步阐明致倦库蚊吸血行为的嗅觉机制奠定基础。同时，本研究还将建立基于致倦库蚊OR蛋白筛选气味物质的方法，为寻找新的致倦库蚊引诱剂和驱避剂候选化合物、干扰蚊虫和宿主之间的联系从而阻断虫媒病的流行提供思路和途径。

利用实时荧光定量PCR技术测定10个ORs在致倦库蚊不同生长发育阶段的表达，以及雌蚊在不同吸血情况下头部的表达。结果显示，CquiOR54在蚊虫卵到蛹以及雄蚊羽化后一周内表达量最低；CquiOR78在雄蚊中特异性表达；CquiOR15、CquiOR54、CquiOR71和CquiOR118在雌蚊饱血与未吸血头部之间的表达存在显著性差异。推测CquiOR15、CquiOR54、CquiOR71、CquiOR78和CquiOR118这五个ORs可能参与了致倦库蚊某些决定性的生理过程。. 通过扩增CquiOR117 和CquiOR5在通过实时荧光定量PCR技术对CquiOR117在致倦库蚊不同生长阶段进行测定，发现CquiOR117的表达量在卵到蛹的阶段明显低于成蚊阶段，雄蚊从羽化后第2天起表达量趋于稳定水平，雌蚊在羽化后第3天表达量达到峰值。利用RNAi技术对CquiOR117和CquiOR5进行干扰，结果显示注射dsRNA 2-4天后，CquiOR117和CquiOR5表达量与对照组都有显著下降；结合蚊虫吸血行为实验发现，CquiOR117和CquiOR5表达量与蚊虫吸血率之间呈显著正相关关系。由此推测CquiOR117和CquiOR5可能在对致倦库蚊的吸血行为有一定的作用。