20E调控桔小实蝇交配后效应的分子机理研究

The oriental fruit fly, Bactrocera dorsalis, is one of the destructive pests for various kinds of fruits and vegetables and shows high fecundity, causing severe economic damage to fruit and vegetable industry. Multiple mating exists in B.dorsalis of both sexes. Mating triggers profound post-mating effects in females, including reducing the rate of remating and stimulating oviposition. The post-mating effects play important parts in population occurrence and reproduction in insects，and can be used as the breakthrough point for pest control. 20-hydroxyecdysone (20E) is the active form of ecdysteriod, being involved in regulation of many physiological processes in insects, such as molting and metamorphasis. Our previous studies indicated that 20E played important roles in regulation of post-mating effects in B.dorsalis. However, the underlying mechanism is unknown. Based on the transcriptome of B.dorsalis previously established by us, this project intends to identify post-mating effect related genes regulated by 20E. Roles of the key genes in regulating reproductive behaviors, such as remating rate and fecundity, will be studied by RNAi. Co-immunoprecipitation assay will be used to identify the interacted proteins for the encoded proteins of key genes. Our study will not only contribute to elucidate the mechanism of post-mating effects in B.dorsalis and lay a foundation for further investigation of the mechanisms of insect reproduction regulation, but also provide theoretical basis for the development of efficient, simple, green and sustainable control strategies of B. dorsalis.

桔小实蝇是重要的果蔬害虫，其寄主范围广、繁殖力强，给果蔬业造成严重的经济损失。桔小实蝇雌雄虫均可多次交配，交配可使雌虫产生"交配后效应"，表现为一段时间内抑制再交配、促进产卵等。该效应在昆虫种群的发生和繁衍中发挥了重要作用，也是控制虫害的重要切入点。20-羟基蜕皮酮（20E）是蜕皮激素的活化形式，参与调控昆虫蜕皮变态等诸多生理过程。我们前期研究发现，20E信号在调控桔小实蝇交配后效应中发挥了重要作用，然而调控机理尚不清楚。本项目拟从我们前期建立的桔小实蝇转录组文库中鉴定受20E调控的交配后效应相关基因；利用RNAi、免疫共沉淀等技术研究关键基因在调控雌虫交配后生殖行为（交配次数和繁殖力）中的功能并鉴定关键基因编码蛋白的互作蛋白。本项目的完成有望揭示20E调控桔小实蝇交配后效应的分子机理，为进一步丰富和完善昆虫生殖调控机制奠定基础，最终为桔小实蝇的高效、简便和可持续绿色防控提供理论基础。

桔小实蝇是重要的世界检疫性果蔬害虫，交配可使雌虫产生表现为一段时间内促进产卵等的交配后效应。交配后效应在昆虫生殖调控中发挥了非常重要的作用，但目前已知的交配后效应相关调控分子非常少，调控机理尚不清楚。本研究通过转录组测序构建了桔小实蝇雌虫交配前后转录组文库，鉴定到了多个交配前后差异表达的基因，GO pathway富集分析结果表明参与刺激应答和免疫信号的基因数量最多，qRT-PCR检测发现多数抗菌肽基因均于交配后24h内在脂肪体中上调表达；此外，20E信号途径基因在雌虫交配前后差异表达；我们进一步鉴定发现了在雌虫交配前后差异表达并且受20E调控的SPR基因，克隆获得了SPR基因全长cDNA序列，SPR属于GPCR受体，具有典型的7次跨膜结构，通过RNAi沉默SPR表达后，雌虫产卵量和后代羽化率显著降低，同时，我们发现成虫存活率和寿命显著低于对照组。本研究不仅为深入认识昆虫生殖调控的分子机制提供了资料，而且为防治害虫提供新思路与新途径，最终为桔小实蝇的高效、简便和可持续绿色防控提供理论基础。

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