水体盐度对中华绒螯蟹卵巢发育和脂质代谢的调控机制研究

The Chinese mitten crab (Eriocheir sinensis) generally migrates downstream to estuaries for reproduction after puberty molt. During this period, gonads of the crabs gradually develop to be mature in the estuaries where they copulate and subsequently spawn. However, it remains unclear how the water salinity influences the ovarian development of E. sinensis. Our previous studies showed water salinity could accelerate ovarian development and significantly affect the lipid metabolism of female E. sinensis. Based on these results, the current project was conducted to investigate the regulatory mechanism of water salinity existing in the ovarian development and lipid metabolism process of E. sinensis. In this project, in vitro tissue culture will be first used to determine the important ions involved in the vitellogenesis and hormone secretion. On this basis, in vivo experiment of different salinity levels/ion concentrations will be applied to make a comprehensive assessment on the effects of water salinity/ions on the ovarian development, hormone secretion, lipid metabolism and relative gene expression in vivo. Furthermore, trans-omics (transcriptomics, proteomics, metabolomics and lipidomics) approach will be employed to screen out the differential expression genes, proteins, hormones and metabolites, which are related to the water salinity regulation of the ovarian development. Through the validation experiment, we plan to establish and verify the regulatory network of the water salinity during the ovarian development and lipid metabolism process, and finally explore the possible mechanism. The expected results will be of great theoretical value and practical significance in the thorough comprehension of ovarian development, spawning migration, artificial propagation and female fattening of E. sinensis.

中华绒螯蟹（以下简称河蟹）生殖蜕壳后通常会洄游到低盐度河口区进行卵巢发育和交配产卵，有关盐度对其卵巢发育的调控机制尚不清楚。本项目前期发现水体盐度可以促进河蟹卵巢发育和影响其脂类代谢。在此基础上，拟系统研究水体盐度对河蟹卵巢发育和脂质代谢的调控机制。项目首先通过组织离体调控实验，筛选出影响河蟹卵黄发生和激素分泌的重要离子；其次通过不同水体盐度/离子浓度下的活体养殖实验，综合评价水体盐度/离子对河蟹卵巢发育、激素分泌、脂质代谢及相关基因表达的影响；进一步采用跨组学（转录组学、蛋白质组学、代谢组学和脂质组学）技术筛选出盐度调控卵巢发育相关的差异表达基因、蛋白、激素和代谢物，结合验证性实验，构建和验证水体盐度对河蟹卵巢发育和脂质代谢的调控网络，探讨其作用机制。预期研究结果对深入理解河蟹卵巢发育、生殖洄游、人工繁殖和雌体育肥等具有重要的理论价值和现实意义。

中华绒螯蟹生殖蜕壳后通常会洄游到低盐度河口区进行卵巢发育和交配产卵，有关盐度对其卵巢发育的调控机制尚不清楚。1、比较了长江野生蟹降海洄游过程中在淡水区与低盐度海水中的肝胰腺和性腺指数变化以及肝胰腺和肌肉中总脂和脂肪酸含量差异。河蟹成体由淡水区洄游至咸水区时，肝胰腺指数及总脂含量均显著降低，咸水区野生亲本已经完成交配和产卵，其肝胰腺和肌肉中的总n-3LC-PUFA百分含量显著升高。2、通过不同含量的Na+、K+、Ca2+、Mg2+培养液离体培养肝胰腺和卵巢组织，评估不同离子对肝胰腺和卵巢中Vg/Vn含量和卵巢发育相关基因表达量的影响。结果表明在离体条件下，Ca2+能够显著促进肝胰腺中Vg合成、提高卵巢发育相关基因如Vg-mRNA、ERR、EcR和RXR基因的表达水平。Mg2+促进肝胰腺中Vg的合成，但对卵巢发育相关基因无显著影响，而Na+和K+对卵黄合成和卵巢发育相关基因表达无显著影响。3、根据离体组织培养结果表明，Ca2+对河蟹卵巢发育相关基因的表达有显著的促进作用。因此本研究在活体条件下，配置两种不同盐度的和不同Ca2+含量的养殖水体，并研究了Mg2+与Ca2+是否存在协同促进卵巢发育作用，结果表明Ca2+能够显著提高卵巢指数、卵径、Vn含量、肝胰腺和卵巢Vg-mRNA和EcR基因表达量，肝胰腺和卵巢中总脂和C22:5n3（DPA）含量、血清甘油三酯和总胆固醇含量的随水体中Ca2+含量增加也呈现上升趋势，推测Ca2+可能是通过影响类固醇激素如蜕皮激素来影响卵巢发育。此外，DPA可能是Ca2+调控卵巢发育和脂质代谢的重要脂肪酸。但Mg2+与Ca2+对促进卵巢发育协同作用不明显。4、设计两个盐度（0‰和6‰）对生殖蜕壳后的雌蟹进行了为期60天的养殖，结合代谢组学探究水体盐度对雌蟹性腺发育、脂肪酸组成和脂质代谢的影响。结果显示6‰组的雌蟹卵巢快速发育，肝胰腺和卵巢总脂含量、花生四烯酸（ARA）以及总LC-PUFA均显著提高，脂肪分解相关酶活力降低。代谢组分析6‰组脂类代谢中亚油酸代谢、鞘脂代谢的中间产物或终产物，如亚油酸、鞘氨醇、磷酸和磷酸乙醇胺含量均在显著下调。综上，水体盐度升高至6‰时，雌蟹降低蛋白质、脂质和碳水化合物分解代谢，增加肝胰腺和卵巢中脂质积累，从而调控卵巢发育。5、研究了活体注射蜕皮激素（20E）对河蟹卵巢发育的影响，结果显示外源20E可显著促进卵巢发育