拟穴青蟹卵巢滤泡细胞的旁分泌作用研究

The paracrine effect in the ovary of vertebrates plays important roles during oocytes development. As to crustaceans, little is known about the paracrine mechanism of ovary. In this project, we plan to carry out following researches on the mud crab Scylla paramamosain, an important culture crustacean: ⑴To screen differentially-expressed genes during different stages of ovarian development by transcriptomics analysis; ⑵To acquire cDNA sequences of significantly regulatory genes by molecular cloning; ⑶To clarify the localization of regulatory genes in follicle cells or oocytes by in-situ hybridization and immunohistochemistry;⑷To expound the regulatory effect of paracrine factors in follicle cells on oocytes development through functional verification,such as RNA interference and gene over-expression. As a result, we tempt to discover a series of genes related to the ovarian development in mud crab, and to reveal the reproductive regulatory mechanism from multi-gene levels. The identification of paracrine factors in follicle cells and the clarification of their paracrine effect will contribute to the comprehension of the mutual interaction between follicle cells and oocytes in mud crab. This project will develop a new research domain in crustacean reproduction, in that it will reveal the mechanism of crustacean ovary development through paracrine way, and also provide scientific supports for the innovation of spawn-inducing technology in crustacean, thus has great theoretical and practical importance.

脊椎动物卵巢的旁分泌作用对卵母细胞的发育具有重要调控作用，迄今甲壳类卵巢旁分泌作用机制尚未见诸报道。本项目以重要养殖甲壳类拟穴青蟹为对象，开展以下研究：(1)通过转录组学分析，筛选不同卵巢发育期差异表达基因；(2)通过分子克隆，获得重要调控基因的cDNA序列；(3)利用原位杂交和免疫组化技术，明确旁分泌因子在滤泡细胞/卵母细胞的定位；(4)开展RNA干扰、基因过表达等技术进行功能验证，确认滤泡细胞中的旁分泌因子对卵母细胞发育的调控作用。本项目拟发掘一批与青蟹卵巢发育相关的基因，从多基因水平较全面地揭示生殖调控机制。有关滤泡细胞旁分泌因子的确定和旁分泌作用的阐明，对了解青蟹滤泡细胞与卵母细胞之间的相互作用将具有重要意义。该项目开辟了甲壳类生殖研究的一个新领域，不仅从旁分泌途径开展甲壳类卵巢发育机制的研究，其结果还可为甲壳类诱导产卵技术的创新提供科学依据，具有重要的理论意义和潜在的应用价值。

脊椎动物卵巢的旁分泌作用对卵母细胞的发育具有重要调控作用，迄今甲壳类卵巢旁分泌作用机制尚未见诸报道。本项目以重要养殖甲壳类拟穴青蟹为对象，开展以下研究：(1)通过转录组学分析，筛选不同卵巢发育期差异表达基因；(2)通过分子克隆，获得重要调控基因的cDNA序列；(3)利用原位杂交和免疫组化技术，明确旁分泌因子在滤泡细胞/卵母细胞的定位；(4)开展RNA干扰、基因过表达等技术进行功能验证，确认滤泡细胞中的旁分泌因子对卵母细胞发育的调控作用。本项目发掘了BMP7、ILP、sNPF、EcR、RXR等青蟹卵巢发育相关的基因，从多基因水平较全面地揭示生殖调控机制。有关滤泡细胞旁分泌因子的确定和旁分泌作用的阐明，对了解青蟹滤泡细胞与卵母细胞之间的相互作用将具有重要意义。该项目开辟了甲壳类生殖研究的一个新领域，不仅从旁分泌途径开展甲壳类卵巢发育机制的研究，其结果还可为甲壳类诱导产卵技术的创新提供科学依据，具有重要的理论意义和潜在的应用价值。