microRNA调控拟穴青蟹卵巢发育的分子机制

The mud crab (Scylla paramamosain) industry yields the highest production among the cultured marine crabs in China. The crab with matured ovary is much higher than ordinary crab in nutrition and price. Therefore, studies on molecular mechanism of ovarian development are very important. Many ovarian development-related genes and miRNAs that are differentially expressed in eyestalks and gonads of different genders have been obtained from our previous projects granted by the National Natural Science Foundation. The miRNAs that are regulators of genes we have identified in our previous projects that includes genes of neuropeptides expressed in eyestalk, and genes that are related to ovarian development, ERK pathway, cell cyclins, and vitellogenesis will be predicted by using bioinformatics tools. The expression of these predicted miRNA and their target genes in eyestalks and ovaries at different developmental stages of ovary will be measured by using real-time PCR. Those miRNAs that are expressed in a negative correlation with their target genes will be chosen for downstream investigation： 1) to test the capability of these miRNAs to regulate their “target genes” by a dual luciferase single plasmid which contains the 3' UTR or mutated 3' UTR of the targeted gene, 2) to exam the expression of protein coded by the “target genes” in in vitro or in vivo system with the inhibition of miRNA to confirm the function of the miRNA. A regulatory network includes miRNA-target gene-GO-pathway will be constructed by using bioinformatics tools which will provide us with a deeper understanding of the molecular mechanism of ovarian development, and lay the foundation for reproduction control in mud crab.

拟穴青蟹是目前我国养殖量最高的海水蟹类。卵巢发育成熟的雌蟹其营养价值和商品价格均远高于普通青蟹，因此其卵巢发育的分子机制一直是研究的重点。本研究在前期获得大量性腺发育相关基因、雌雄眼柄和性腺差异表达miRNA的基础上，将采用生物信息学的方法寻找调控眼柄神经肽基因、ERK通路基因、细胞周期蛋白基因和卵黄发生关键基因的miRNA。定量PCR筛选表达水平的变化与卵巢发育相关基因呈负相关的miRNA。通过1）构建这些miRNA靶基因的3'UTR双荧光素酶报告基因载体和突变型载体转染细胞系，通过双荧光素酶报告系统酶活性变化以验证miRNA对靶基因的调控能力，2）离体或在体抑制miRNA后检测靶基因编码蛋白的水平以确认这些miRNA对其靶基因的调控作用。进一步利用生物信息学构建miRNA-靶基因-GO-pathway的调控网络，进一步探讨拟穴青蟹卵巢发育的分子机理，为其生殖过程的人工调控提供新的思路。

拟穴青蟹是我国最重要的海水养殖蟹类之一，其卵子质量的好坏与其孵化率、幼体的质量和成活率等密切相关，卵巢发育成熟的雌蟹其价格和营养远高于普通青蟹。因此青蟹卵巢发育机制的研究既具重要的理论价值也有重要的现实意义。.越来越多的证据表明miRNA参与动物的生殖调控。本课题以眼柄神经肽-ERK-Vtg通路，ERK-细胞周期-卵细胞成熟为主线，选取已获得完整3’-UTR的眼柄神经肽基因，ERK通路基因，细胞周期基因和卵黄发生关键基因，在课题组已完成雌雄眼柄、肝胰腺和性腺的miRNA组测序的基础上，进行了以下研究：调控目的基因的miRNA预测和筛选，候选miRNA在卵巢发育过程中的表达模式分析，miRNA的功能分析：（a）通过构建包含卵巢发育相关基因的3'UTR 的双荧光素酶报告基因载体，并通过人工合成miRNA mimics或miRNA inhibitor，共转染HEK293T细胞系，根据荧光值的变化来确认miRNA是否调控靶基因的表达，初步确认miRNA的功能。（b）通过位点定向突变构建3’-UTR突变型荧光素酶载体，检测报告基因的表达，以进一步确认miRNA功能。（c）通过miRNA antagomir或miRNA agomir进行在体实验确认miRNA对基因表达的影响。通过上述研究，我们发现1）在精巢和卵巢中存在108个差异表达的miRNAs。特别是miR-9c,miR-279e,miR-263a,miR-263b,miR-9和miR-34对许多性腺发育相关的基因可能有调控作用，且在卵巢发育过程中差异表达；2）miR-277可以通过调控青蟹眼柄内VIH基因的表达影响vtg的表达，进而影响卵巢的发育；3）miR-34可以通过调控青蟹眼柄内MIH基因、CHH基因、EcR基因和FAMeT基因的表达进而参与生殖调控；4）青蟹卵巢和肝胰腺组织中miR-9c对ERK2和MEK2基因表达具有抑制作用，miR-263a对ERK2、MEK2和Rap-1b基因的表达具有抑制。5）miR-9c和miR-263a可以通过调控细胞周期相关基因cyclins和CDKs的表达进一步影响vtg的表达进而影响卵巢发育。6）miR-34可以通过调控青蟹vtg和vtgR的表达参与卵巢的发育。这些研究成果使我们在理论上更加深入地了解了拟穴青蟹性腺发育的调控机制，在生产实践为建立新的生殖调控技术提供了新的思路。