华溪蟹卵子发生相关调控基因的筛选与鉴定及作用研究

Oogenesis is regulated by the genetic factors, which determines the quality of embryo. In our previous study, we have confirmed the significant differences of oogenesis between Sinopotamon and other crabs, including that the volume of oocyte in Sinopotamon is 10 to 20 times larger than that of its closely related species, and its embryo performs the direct development. It is suggested that the freshwater crab, Sinopotamon has the unique regulation mechanism of oogenesis. However, the mechanisms of oogenesis of Sinopotamon remain unknown. Moreover, we have explored the histological and cytological changes of oogenesis and ovary development, and constructed the cDNA library of ovary as well as identified the reproductive marker vasa in Sinopotamon henanense. Based on these results, this project will identify the differentially expressed genes (DEGs) specific to ovary and related to the oogenesis of S. henanense using comparative transcriptome analysis. Using vasa as a molecular marker, we will further identify the DEGs specially expressed in oocyte by the two colors of fluorescent in situ hybridization, and select the new or key regulation molecules. After we clone the key regulation genes, we will detect their dynamic expression and distribution in oocyte and reveal their spatial and temporal expression patterns. Furthermore, after knockdown the key regulation genes, we will compare and analyze the changes of expression and localization of their and the upstream and downstream genes, explore their effects on oogenesis, and reveal their functions. Finally, we will illustrate the molecular mechanisms of oogenesis in Sinopotamon. These results will expand the knowledge of reproduction and development especially the oogenesis in crustacean, and will provide the scientific basis for mastering the reproductive regulation mechanisms of crustaceans.

动物卵子发生过程受遗传因素和基因调控的影响，也决定了胚胎的质量。前期研究已证实华溪蟹卵子发生与其他蟹类显著不同，卵子的大小也要比其它同类大10-20倍，胚胎进行直接发育，这些预示着华溪蟹的卵子发生存在独特的调控方式，但相关机制目前尚不清楚。基于我们已完成的河南华溪蟹卵巢发育和卵子发生的形态观察、卵巢cDNA文库的构建、生殖标记基因vasa的鉴定等工作，本项目拟采用比较转录组等方法，首先筛查在河南华溪蟹卵巢中特异表达、且与卵子发生相关的差异表达基因；然后结合双色荧光原位杂交，鉴定出在卵母细胞中特异表达的新的或关键调控基因，并测试它们的时空表达模式；再进一步利用基因敲降技术，分析比较所鉴定的基因对卵母细胞发育和成熟的影响及其作用方式，以阐明华溪蟹卵子发生的分子机制。研究结果不但能拓展甲壳动物生殖发育、尤其是卵子发生的理论知识，而且为掌握甲壳动物的生殖调控机制提供科学依据。

卵子发生的正常与否决定着受精卵的质量和胚胎发育的进程。卵子发生是一个复杂的基因调控过程，期间表达和积累的大分子物质在卵子生长、成熟、受精及早期胚胎发育等过程中发挥着至关重要的作用。前期研究已证实华溪蟹卵子发生与其他蟹类显著不同，且胚胎进行直接发育，这些预示着华溪蟹的卵子发生存在独特的调控方式，但相关机制目前尚不清楚。本项目采用比较转录组的方法，首次建立了河南华溪蟹（Sinopotamon henanense）卵巢转录组数据库，筛查了河南华溪蟹卵巢中表达的差基因，分析了与卵子发生相关的差异表达基因。在此基础上，分别克隆获得了vasa、nanos、piwi 3个关键的生殖调控分子，揭示了其时空表达模式，明确了VASA蛋白在卵母细胞中由胞质到胞核的动态分布，探讨了其在河南华溪蟹卵子发生过程中的生物学功能。进一步，追踪了镉暴露后生殖基因vasa RNA和蛋白在卵母细胞中的含量和分布状态，检测了镉暴露对vasa、nanos、piwi基因表达的影响。结合基因敲降技术，分析了3个生殖基因作用关系，结果表明，敲降vasa基因导致nanos和piwi基因显著下调表达，但同时干扰vasa基因和镉暴露，则显著诱导了nanos基因的表达，而抑制了piwi基因的表达。研究确定了镉暴露对生殖调控基因的毒性效应，分析了镉对河南华溪蟹生殖毒性的分子机制。本项目对华溪蟹卵子发生相关调控基因的筛选、鉴定及其作用机制的研究，为深入探讨甲壳动物不同发育方式的分子调控机制提供了理论基础，为蟹类水产养殖和动物资源保护与开发中如何提高卵子质量提供了科学依据，同时为环境毒理学研究中，重金属胁迫的生殖毒性效应机制研究提供了研究基础，为水环境监测中提供了标记分子。