20E和Insulin调控桔小实蝇雌虫性成熟分子机理研究

The oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae) is one of the most important pests for various kinds of fruits and vegetables.It is difficult for us to control this pest. Females can't reach sexual maturation to mate and lay eggs after emergence, until they further develop for several days. Therefore, the process to reach sexual maturation is considered as the important breakthrough point for pest control. Both 20-hydroxyecdysone (20E) and insulin are the most important endocrine hormones for insects, playing very important roles in insect reproductive process. Our results showed that several key transcription factors were significantly differentially expressed between the newly emerged females and the sexually mature females, such as EcR、FOXO, etc. This data suggest that both signal pathways are involved in regulating sexual maturation. However, the underlying mechanism and the possible crosstalk between these two signaling pathways are unknown. This project intends to identify more genes regulated by 20E and insulin participating in the process of female sexual maturation using transcriptome sequencing. Roles of these genes in regulating sexual maturation will be studied by RNAi. The yeast two-hybrid system will be used to identify the interacted proteins for the encoded proteins of key genes. Co-immunoprecipitation will also be used to to analyze their interaction. Our study will not only illustrate the mechanism of sexual maturation regulated by 20E and insulin signaling 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，防治困难。其成虫羽化后必须经历一段时间才能达到性成熟并交配产卵，因而性成熟过程也是控制虫害的重要切入点。20-羟基蜕皮酮(20E)和胰岛素(Insulin) 是昆虫体内重要的内分泌激素，参与调控昆虫生殖发育等诸多生理过程。我们的研究显示，20E和Insulin信号途径中的关键转录因子EcR、FOXO等基因在桔小实蝇雌虫性成熟前后差异表达，表明两种信号参与调控桔小实蝇雌虫性成熟。然而其调控机理及两种信号途径交联方式尚不清楚。本项目拟通过转录组表达差异分析，鉴定受20E和Insulin调控的性成熟相关基因；利用RNAi、酵母双杂交、免疫共沉淀等技术研究关键基因在调控雌虫性成熟中的功能及其编码蛋白的互作蛋白。本项目将揭示20E和Insulin调控桔小实蝇性成熟的分子机理，为进一步丰富和完善昆虫生殖调控机制提供新线索，最终为桔小实蝇高效、简便和可持续绿色防控提供理论基础。

多数昆虫的成虫羽化后需要补充营养才能达到性成熟、从而促进交配产卵，但目前已知的性成熟调控基因非常少，调控机理尚不清楚。桔小实蝇是重要的果蔬害虫，防治困难。其成虫羽化后必须经历一段时间才能达到性成熟并交配产卵，因而性成熟过程也是控制虫害的重要切入点。本项目建立了桔小实蝇雌虫初羽化与性成熟时期转录组文库，DGE分析鉴定了多个差异表达基因；qRT-PCR进行了验证，并检测了20E和Insulin对差异表达基因的调控模式。RNAi沉默20E信号通路的重要转录因子EcR、USP和Insulin信号通路重要转录因子InR、Foxo，桔小实蝇产卵量显著减少，卵黄蛋白原基因Vg表达量明显下调，表明20E和Insulin信号通路通过影响雌虫卵黄沉积和产卵量调控其生殖。鉴定到在桔小实蝇雌虫性成熟时期高表达的Rab40基因；注射20E和Insulin后Rab40表达量明显上调；RNAi干扰BdRab40后，雌成虫的卵巢组织发育迟缓或不完全，并且总产卵量及日均产卵量都显著降低；卵黄蛋白原的表达量明显下调。此外，从桔小实蝇成虫转录组中鉴定得到了6个在初羽化与性成熟时期差异表达的COP9信号复合体亚单位基因csn2、csn3、csn4、csn5、csn6、csn7；时空表达模式结果表明，csn3、csn5在性成熟时期及卵巢组织高表达；沉默csn5基因后，卵巢发育明显迟缓，并且产卵量显著下调；蜕皮激素合成通路与20E信号途径关键转录因子表达量显著下调；酵母双杂交技术筛选到CSN5互作蛋白Vg1，pull down验证发现CSN5与Vg1存在互作。另外，鉴定到在性成熟时期的卵巢组织中高表达的蛋白酶体β3亚基类似物基因 pts，实时荧光定量PCR和免疫印迹技术发现其在性成熟时期的卵巢组织高表达，免疫组化表明pts蛋白主要定位于卵巢组织滤泡细胞和滋养细胞的细胞质中， RNAi沉默该基因后，约有1/2的雌虫卵巢发育迟缓，脂肪体中的卵黄原蛋白基因Vg1、Vg2及卵黄原蛋白Vg1、Vg2表达量显著下调。以上结果表明，Rab40、csn5、pts基因可能通过影响桔小实蝇卵黄原蛋白合成与卵黄沉积作用参与调控卵巢发育与产卵量，进而影响雌虫的生殖发育。本项目揭示了20E 和Insulin调控桔小实蝇性成熟的分子机理，为进一步丰富和完善昆虫生殖调控机制提供新线索，最终为桔小实蝇高效、简便和可持续绿色防控提供理论基础。

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