甲基法尼酯在三疣梭子蟹卵巢发育和蜕皮过程中的生理作用

The swimming crab, Portunus trituberculatus, is one of the major species for marine aquaculture in China. The physiological function of methyl farnesoate (MF) is a hot research topic of crustacean endocrinology. The roles of MF in the regulation of ovarian development and molting may vary among species, developmental stage and MF level, and more studies are needed. The changes of gene expression levels of molt-inhibiting hormone (MIH) and hemolymph levels of ecdysteroid during molting and ovarian development in P. trituberculatus have been elucidated. A full-length farnesoic acid -O-methyl transferase (FAMeT) cDNA from P. trituberculatus has been cloned by the reverse transcription-polymerase chain reaction (RT-PCR) and the rapid amplification of cDNA ends (RACE). And a method based on capillary gas chromatography-mass spectrometry has been established for determination of hemolymph levels of MF. On the basis of these results, this project will aim to: (1) detect the changes of hemolymph levels of MF, MIH and mandibular organ-inhibiting hormone (MOIH) during ovarian development and molting; (2) analyze the changes of gene expression levels of FAMeT, MOIH and retinoid X receptor (RXR) during ovarian development and molting; (3) detect the changes of gene expression levels of vitellogenin (Vg) and hemolymph levels of Vg during ovarian development; (4) investigate further the regulation mode of MF on ovarian development and molting by biological activity assays of in vitro culture and in vivo injection. (5) elucidate the functions of FAMeT, MOIH and RXR in the process of ovarian development and molting by the dsRNA-mediated RNA interference (RNAi) technique. To carry out this study will help to clarify the endocrine regulation mechanism of ovarian development and molting in P. trituberculatus, and enrich the crustacean endocrinology. Additionally, it will provide theoretical guidance for the artificial regulation of ovarian development and molting of P. trituberculatus.

三疣梭子蟹是我国海水人工增养殖的主要种类之一。甲基法尼酯（MF）对甲壳动物卵巢发育和蜕皮的调控作用可能因种、发育阶段和MF剂量而异，有必要深入研究。在已探明三疣梭子蟹卵巢发育和蜕皮周期中MIH基因表达水平及蜕皮类固醇浓度变化规律、克隆FAMeT全长cDNA序列和建立MF浓度GC/MS检测法的基础上，本项目拟分析卵巢发育和蜕皮周期中血淋巴MF、MIH和MOIH浓度变化，FAMeT、MOIH和RXR基因表达水平变化，卵巢发育过程中Vg基因表达水平和血淋巴Vg浓度变化；通过离体培养和活体注射相结合的活性分析进一步揭示MF对卵巢发育和蜕皮的调控模式；通过RNA干扰分析FAMeT、MOIH和RXR等基因在卵巢发育和蜕皮中的功能。本项研究的开展，将有助于阐明甲壳动物卵巢发育和蜕皮的调控机制以及MF合成和作用的机理，丰富甲壳动物内分泌学理论；将为实现三疣梭子蟹卵巢发育和蜕皮的人工调控提供理论指导。

甲基法尼酯（MF）的生理功能及作用机理是甲壳动物内分泌学研究的热点，其研究成果有助于探索人工调控虾蟹类卵巢发育和蜕皮的新方法。采用GC/MS法分析了三疣梭子蟹卵巢发育过程和蜕皮周期中血淋巴MF浓度变化，发现血淋巴MF含量的显著上升能刺激卵黄蛋白原（Vg）基因表达量的显著上升，进而加速卵巢发育进程；MF还可以通过刺激YO合成、分泌蜕皮激素来调控蜕皮。采用qPCR技术进行了HMGR、FAMeT、JHAMT、AACT、FPS等MF合成酶基因的表达水平分析，进一步证实大颚器是甲壳动物合成MF的唯一器官；在卵巢发育和蜕皮过程中大颚器HMGR、FAMeT和JHAMT基因的表达水平变化与血淋巴MF浓度变化趋势大体一致。利用dsRNA介导的RNAi进一步研究了JHAMT和FAMeT在MF生物合成中的作用，发现JHAMT和FAMeT都参与了三疣梭子蟹MF的合成，但JHAMT可能比FAMeT具有更高的底物特异性。以转录组数据库为基础，克隆得到了保幼激素环氧水解酶(JHEH)基因和2个类保幼激素羧酸酯酶（JHE-like CXEs）基因的cDNA序列，发现JHEH和CXE1在肝胰腺中强烈表达，CXE2主要在肝胰腺和卵巢中表达，CXEs在肝胰腺中的表达受到MF水平的调控，这为CXEs具有降解MF的功能提供了重要的证据。克隆获得了三疣梭子蟹候选MF受体Met基因和可能的MF信号早期响应基因Kr-h1的全长cDNA序列，离体培养和活体注射实验显示，MF能促进肝胰腺中Met和Vg基因表达水平的上调，肝胰腺是Vg的主要合成组织，因此推测Met作为MF的受体参与MF诱导的肝胰腺Vg基因转录激活过程。这些研究成果不仅在阐明MF对卵巢发育和蜕皮的调控作用、机理，丰富甲壳动物内分泌学知识方面具有重要的理论意义；而且能为建立基于MF的有效的人工调控虾蟹类卵巢发育和蜕皮的新方法提供理论支撑。