已有的研究表明,视黄醇结合蛋白(rbp)除了影响甲壳动物的体色、体壳外，对它们的性腺发育和生殖也有重要作用。为了更好地了解此类蛋白分子对三疣梭子蟹卵巢发育的调控作用,本项目在克隆三疣梭子蟹的rbp分子（pt-rbp）全长cDNA序列的基础上，利用定量PCR和Western blotting全面分析了其时空表达特征；利用RNAi和投喂VA分别研究pt-rbp的下调和上调表达对卵巢发育的作用；最后通过检测几种激素的分泌和激素受体的表达情况，初步揭示其通过内分泌系统的调控机理。研究结果有助于揭示三疣梭子蟹卵巢发育的调控机理，有助于促进其人工育苗和培育优良的繁育亲本。

In addition to its essential role in crustacean shell color, retinol binding protein (rbp) has been reported to be also critical in gonad maturation, reproduction and development. In order to discover the regulation by which rbp functions in the vitellogenesis of swimming crab , Portunus trituberculatus, the full-length cDNA of rbp from Portunus trituberculatus (pt-rbp) has been cloned. In the project, the expression patterns of pt-rbp would be characterized using Real-time PCR and Western blotting. Furthermore, the up-regulation and down-regulation of pt-rbp would be induced by RNAi of pt-rbp and addition of VA in dairy feeding, respectively.Therefore, the effects of up and down-regulation of pt-rbp on the vitellogenesis of P. Trituberculatus would be clarified. Finally, the roles of pt-rbp on some hormone produced and expression of hormone receptors would be investigated. The results contribute to our understanding of the mechanism of vitellogenesis of P. trituberculatus, and are helpful for artificial larva breeding and parent with higher reproduction capacity breeding.

本研究完成了三疣梭子蟹lipocalin家族的两个成员pt-rbp和pt-fabp全长cDNA序列的解析和生物信息学分析。这两个蛋白对于维生素A 类物质以及其他亲脂性物质的转运具有重要作用。通过RT-PCR分析了三疣梭子蟹不同组织、不同发育阶段pt-fabp和pt-rbp的转录水平。利用原核表达和ITC研究，发现pt-rbp与视黄酸和脂肪酸分子均可能有结合特异性。通过喂食VA有效地增加了pt-rbp的表达，进而通过RT-PCR分析几种基因的表达情况。结果发现视黄酸信号通路的下游响应元件RXR、EcR的表达均有增加，证明pt-rbp在视黄酸信号通路中具有承前启后的作用。利用GC-MS 和LC-MS分析了三疣梭子蟹不同组织中脂肪酸组成和视黄酸的含量，并与pt-fabp和pt-rbp的表达建立初步的联系。研究发现pt-fabp在雌雄生殖腺中具有较高的表达量，与脂肪酸组成和视黄酸含量具有一定的相关性。通过本课题的研究建立了三疣梭子蟹组织中视黄酸的提取和LC-MS的检测方法。视黄酸、VA、虾青素等亲脂分子在甲壳动物的体色、免疫防御、生长发育、性腺发育均具有重要作用。本课题的研究为阐明这类分子的真正生理作用，以及它们通过视黄酸信号通路的调控方式提供了数据和依据。