Foxl2在三疣梭子蟹卵巢发育中的作用及其机制研究

Forkhead box L2（Foxl2）is a transcription factor, which plays a critical role in regulating the ovarian development of animals. In our previous study, we have obtained the partial cDNA sequences of Foxl2 and its target genes of the swimming crab, Portunus trituberculatus. The current project will investigate the role and mechanism of Foxl2 in the ovarian development of female P. trituberculatus. Firstly, we will clone and sequence Foxl2 and its target genes, and determine the expression pattern of Foxl2 at mRNA and protein levels during the ovarian development. Secondly, to determine the role of Foxl2 in P.trituberculatus ovarian development, we will examine the effects of knocking down Foxl2 expression on the ovarian development. And then, we will verify the interaction between Foxl2 and its target genes, and clarify the correlation between the expression of Foxl2 and its target genes during the ovarian development. Finally, we will elucidate the regulatory mechanism of Foxl2 in P.trituberculatus ovarian development, by analyzing how Foxl2 knockdown affects the expression of its target genes. This research focuses on an important transcription factor to find a new way of investigating the molecular mechanisms of P.trituberculatus ovarian development. The expected outcomes of this research would not only improve our understanding of the molecular mechanisms of the ovarian development of P.trituberculatus and other crustacea, but also provide the basic information for studying the ovarian hypoplasia in pond-reared P.trituberculatus.

叉头框蛋白L2（Forkhead box L2，Foxl2）是在卵巢发育中发挥重要调控作用的一种转录因子。本项目在已获得的三疣梭子蟹Foxl2及其下游靶基因序列片段的基础上，克隆这些基因的全长序列；从mRNA和蛋白水平分析Foxl2在三疣梭子蟹卵巢发育过程中的表达特征；通过检测RNA干扰Foxl2表达对卵巢发育的影响，分析Foxl2在卵巢发育中的作用；明确Foxl2与其靶基因的相互作用，分析Foxl2与其靶基因在三疣梭子蟹卵巢发育过程中表达模式的相关性，并研究干扰Foxl2对其靶基因表达的影响，从而阐明Foxl2在卵巢发育中发挥作用的机制。本项目针对转录因子开展研究，尝试寻找三疣梭子蟹卵巢发育分子调控机制研究的新切入点。预期的研究结果对于深入理解三疣梭子蟹等甲壳动物卵巢发育的分子调控机制具有重要意义，为解决池塘养殖三疣梭子蟹卵巢发育不良的问题奠定理论基础。

叉头框蛋白L2（Forkhead box protein L2，Foxl2）在脊椎动物卵巢发育中发挥重要的调控作用，但其在甲壳动物卵巢发育中的功能尚不明确。本项目以重要海水经济甲壳类三疣梭子蟹为实验对象，探索了Foxl2基因在三疣梭子蟹卵巢发育中的作用及其机制。主要结果如下：. 构建了三疣梭子蟹卵巢组织转录组数据库，并在数据库基因片段基础上克隆了三疣梭子蟹Foxl2（PtFoxl2）cDNA全长序列，对PtFoxl2进行了原核重组表达，制备多克隆抗体。利用real-time PCR、原位杂交和western blot从转录和翻译水平对PtFoxl2的时空表达特征进行了研究，结果显示其在卵巢组织中表达量最高，主要定位于卵黄合成前卵母细胞的胞质和滤泡细胞中；此外，PtFoxl2在卵巢发育不同时期的表达差异显著，提示其可能参与了三疣梭子蟹卵巢发育调控。. 为进一步解析PtFoxl2的功能，分析了干扰PtFoxl2表达对三疣梭子蟹卵巢基因表达的影响。结果显示，干扰PtFoxl2表达后，PtGnRHR和PtSTAR基因的表达未出现显著变化，表明PtFoxl2的下游靶基因与脊椎动物不同，不参与PtGnRHR和PtSTAR基因的表达调控；干扰PtFoxl2表达后，PtVtg和PtCyclin H基因的表达显著上调，PtCyclin B和PtCDK7基因的表达显著下调，提示PtFoxl2参与了三疣梭子蟹内源性卵黄合成的调控，可能还与卵巢发育过程中的细胞周期调控有关。. 此外，还开展了PtFoxl2表达调控研究。发现切除眼柄和注射雌激素能够诱导三疣梭子蟹卵巢PtFoxl2的表达，表明PtFoxl2的表达受眼柄神经内分泌调控，其表达与雌激素密切相关；通过三疣梭子蟹卵巢microRNA表达谱分析，筛选了与PtFoxl2表达调控相关的microRNA，并在细胞水平进行了验证，揭示了mir-466i对PtFoxl2表达的调控作用。. 本项目的研究结果有助于深入了解三疣梭子蟹卵巢发育的分子调控机制，而且为开发甲壳动物生殖调控新技术提供了理论参考。