除虫菊素对白纹伊蚊驱避作用的分子机制研究

Pyrethrum is a mixture of six insecticidally active pyrethrin, exhibiting insecticidal toxicity and repellency to pests, the target of pyrethrum repellency is olfactory receptor, however, the mechanism of pyethrum repellency is still elusive. In this proposal, we propose olfactory receptor is another target of pyrethrum, and the objective is to reveal the the mode of action of pyrethrum repellency at molecular level based on Aedes albopictus. In this proposal, six components are isolated and purified by Semi preparative liquid chromatography and the mechanism of pyrethrum repellency is explored on component which shows highest repellency to mosquitoes selected by arm in cage assay and EAG assay. Analysizing transcriptome and expression profiling of Aedes albopictus after exposured to pyrethrin and cloning the olfactory receptors of Aedes albopictus whose expression level varied greatly. The olfactory receptors of Aedes albopictus are expressed in the Xenopus oocyte and two-electrode voltage clamping is used to select the candidate pyrethrum-responsive odorant receptors, futher we will knock out the target olfactory receptor gene using CRISPR to make the transgenic mosquito, then perform arm in cage assay and EAG assay to examine the behavior activity of transgenic mosquito to components of pyrethrum. Thus the target of odorant receptor and the mechanism of pyrethrum repellency is revealed. The expected findings from this project will improve our understanding of the mechanism of pyrethrum repellency at the molecular level, and provide valuable information for future screening and development of new and more potent repellents against mosquitoes and other insect disease vectors. At the same time, this research will provide the model to explore the mechanism of repellents against other pests.

天然除虫菊素是六种除虫菊素组成的混合物，对害虫具有毒杀性和驱避性，其驱避作用的靶标为嗅觉受体，但是其作用机理尚未明确，本项目以白纹伊蚊为研究对象，旨在从分子水平揭示除虫菊素驱避作用的机制。本研究采用半制备液相色谱分离制备六种除虫菊素，通过行为试验和EAG试验筛选驱避作用最强的除虫菊素，用于除虫菊素驱避作用机理的研究。将除虫菊素熏蒸处理后的白纹伊蚊触角进行转录组测序，分析并克隆表达量变化明显的嗅觉受体基因。将嗅觉受体基因表达于爪蟾卵母细胞，通过双电极电压钳技术筛选除虫菊素的候选靶标嗅觉受体，进一步采用CRISPR技术敲除靶标嗅觉受体基因，驱避试验和EAG试验验证嗅觉受体基因的功能，从而定位除虫菊素的靶标嗅觉受体，揭示除虫菊素作用的分子机理。预期研究成果能使我们更好地从分子水平理解除虫菊素驱避性的作用机制，并为开发高效的蚊子驱避剂提供理论基础。同时，也为其他害虫驱避剂的研究提供试验模型。

植物源杀虫剂天然除虫菊素是六种除虫菊素组成的混合物，对蚊虫不仅具有毒杀作用，还具有驱避作用。本项目研究发现天然除虫菊素对白纹伊蚊的驱避效力与传统驱避剂避蚊胺相当，且白纹伊蚊触角能感受天然除虫菊素。利用半制备液相色谱、高效液相色谱和高分辨串联质谱等手段，分离、制备和鉴定得到六种纯度大于98%的除虫菊素，并发现除虫菊素I、除虫菊素II、瓜叶菊素II和茉酮菊素II对白纹伊蚊具有驱避作用，可作为驱避剂。对蚊虫嗅觉受体进行功能分析，发现冈比亚按蚊OR31、埃及伊蚊OR31均在天然除虫菊素的识别中起重要作用，而白纹伊蚊OR31则无法识别天然除虫菊素。进一步通过单细胞记录技术发现白纹伊蚊雌蚊触角中一类sst亚型的嗅觉感器感知天然除虫菊素。此外，对若干白纹伊蚊嗅觉受体进行功能鉴定，明确多个受体对人类挥发性气味及其他驱避剂茴香酮的识别作用。本项目研究结果使我们更好地从分子水平理解除虫菊素驱避性的作用机制，并为开发高效的蚊子驱避剂提供理论基础。