柑桔全爪螨蜕皮激素活性物质鉴定及与受体EcR-RXR1/RXR2结合模式研究

Based on the regulating mechanisms of pest insect or mite grwoth and development to design specific control agents is the hot research filed currently. In this project, we focus on the key question: the identification of ecdysone in spider mites and its combination model with the receptor, through the study from citrus red mites (Panonychus citri) being one of the world-wide destructive pests on citrus. On the basis of our previous study we found that spider mites and insects could use different ecdysone in control of moulting, and also screened the expression dynamics during moulting of several canonical genes involved in ecdysone biosynthesis and signaling, in current project, we will comprehensively use the state-of-art methods and technologies including: the comparison study of genomes and transcriptomes, RNA interference, fluorescent in situ hybridization, immunohistochemical analysis, co-immunoprecipitation, cell-based reporting system, and HPLC-enzyme immunoassay and liquid chromatography/mass spectrometry, for analyzing the process of Halloween-P450 based ecdysone synthesis and moulting regulation, in order to elucidate the ecdysone and the synthesized tissue in citrus red mites, and the mite ecdysone induced combination model between PcEcR with two ligands PcRXR1 and PcRXR2 in together with revealing the interaction with later moulting signaling. This project is aim to provide the molecular mechanism on the biosynthesis and signaling the ecdysone in regulating the growth and development of P. citri. Moreover, our project will improve our current understanding on the general theory in the growth and development of spider mites, and provide new insights for new acaricides development in targeting key factors in control of growth and development of spider mites.

基于解析害虫（螨）生长发育调控机理设计特异控制剂是当前研究的热点领域。本项目以柑桔全爪螨为对象，针对叶螨蜕皮激素鉴定及与其受体结合模式这一关键科学问题，在前期研究发现叶螨与昆虫蜕皮激素活性物质存在差异并初步解析该合成与信号通路上几个基因表达模式的基础上，综合运用比较基因组和转录组学、RNA干扰、原位杂交、免疫组化、免疫共沉淀、异源表达报告系统及色谱分析等技术和方法，解析柑桔全爪螨Halloween-P450基因介导蜕皮激素合成与作用过程，明确柑桔全爪螨蜕皮激素活性物质及合成组织、蜕皮激素诱导的受体PcEcR与PcRXR1和PcRXR2在蜕皮过程中的结合模式及与下游信号通路的互作方式，揭示蜕皮激素合成与信号通路调控柑桔全爪螨生长发育的分子机制。研究结果在理论上将深化对叶螨生长发育调控分子机理的的认识，在实践上将对以叶螨生长发育过程中关键调控因子为靶标设计特异的新型杀螨剂具有重要的指导意义。

本项目以柑桔重要害螨柑桔全爪螨为研究对象，针对叶螨蜕皮激素鉴定及与其受体结合模式这一关键科学问题，在前期研究发现叶螨与昆虫蜕皮激素活性物质存在差异并初步解析该合成与信号通路上几个基因表达模式的基础上，利用基因组和转录组测序联合分析技术对柑桔全爪螨幼螨至成螨生长发育过程蜕皮前后样品进行转录测序，发现大部分差异基因与激素合成、蛋白质合成与吸收、以及营养物质合成和运输有关。基于柑桔全爪螨不同发育阶段转录组和全基因组数据联合分析，共获得15条蜕皮激素合成和信号通路同源基因。利用RNA干扰技术和拯救机制，发现Ponasterone A可以拯救dsPcSpo对柑桔全爪螨蜕皮的抑制作用，证实Ponasterone A是柑桔全爪螨蜕皮激素的活性物质。利用原位杂交技术，明确了中枢神经块是柑桔全爪螨蜕皮激素合成和信号传导作用组织。综合运用RNA干扰和比较转录组学技术，分析了蜕皮激素受体PcEcR与PcRXR1和PcRXR2在蜕皮过程中的结合模式及与下游信号通路的互作方式，揭示蜕皮激素合成与信号通路调控柑桔全爪螨生长发育的分子机制。研究结果在理论上将深化对叶螨生长发育调控分子机理的的认识，在实践上将对以叶螨生长发育过程中关键调控因子为靶标设计特异的新型杀螨剂具有重要的指导意义。