三疣梭子蟹JNK信号通路参与卵黄积累过程中生殖调控的分子机制

The accumulation of vitellin in crustacean is directly related to the hatchability rate of fertilized eggs, which is the key to the breeding work of cultivation industry. According to the report, The JNK signaling pathway was involved in the regulation of many mammals reproduction; however, its role in marine crustaceans still remain unclear. In our laboratory, we conducted a series of studies on ovarian development of Portunus trituberculatus and established a transcriptome library of ovarian development in every period. According to the comparison of differently expressed genes involved in KEGG pathways from mature and immature ovary transcriptome, we found that JNK pathway might participate in the regulation of ovarian development in Portunus trituberculatus. This subject, based on laboratory research, intends to clone the key gene cDNA such as JNK, c-Jun, c-Fos and so on in JNK pathway combining with obtained ESTs and RACE techniques, analyze the expression of vitellogenin with them, and verify whether c-Jun interacts with c-Fos to form complex AP-1 to participate in downstream regulation through IP technology. Then, respectively adopting the methods of the RNAi technique and adding activator and inhibitor of JNK signal specificity to research the influence on vitellogenin synthesis and egg development. This study will illustrate the molecular mechanisms in the process of accumulation of vitellin in Portunus trituberculatus, and provide the theoretical basis for the development of artificial breeding control technology

卵黄积累直接关系到甲壳动物子代孵化，是养殖业繁育工作的关键。据报道，JNK信号通路参与多种哺乳动物生殖相关的调控，但其在海洋甲壳动物中的作用尚未报道。本实验室构建了三疣梭子蟹卵巢各时期的转录组文库，比较成熟与未成熟卵巢转录组差异基因所参与的KEGG通路发现，JNK信号通路参与了三疣梭子蟹卵巢发育的调控。本课题拟在实验室研究基础上，结合已获的EST，通过RACE技术克隆JNK信号通路关键基因JNK及c-Jun等cDNA全长，分析其与卵黄蛋白原的表达变化特征，并利用IP技术验证c-Jun与c-Fos是否互作形成复合体AP-1参与下游调控；采用RNAi技术、添加JNK信号特异性激活剂及抑制剂，研究JNK信号激活或阻断对卵黄蛋白原合成及卵发育的影响。本研究将阐明三疣梭子蟹JNK信号通路调控卵黄积累过程中生殖调控的分子机制，为促进人工繁育调控技术的发展提供理论基础。

三疣梭子蟹是我国沿海重要的人工增养殖品种，繁育是水产养殖重要的一环，卵黄积累程度与苗种质量密切相关。本研究克隆了三疣梭子蟹JNK信号通路的关键基因jnk和c-fos基因全长cDNA序列，jnk cDNA全长为2094 bp，ORF为1266 bp，编码421个氨基酸，具有TPY磷酸化位点的S_TKC保守结构域。c-fos cDNA全长为2829 bp，ORF为1497 bp，编码498个氨基酸。卵巢发育过程中JNK信号通路相关基因和Vg基因在卵巢和肝胰腺组织内均表达。肝胰腺组织中，jnk-mRNA和Vg-mRNA在III期的相对表达量最高，c-jun-mRNA和c-fos-mRNA在IV期的相对表达量最低；卵巢组织中，jnk-mRNA在IV期和V期的相对表达量最高，c-jun-mRNA和c-fos-mRNA在III期的相对表达量最低，而Vg-mRNA的表达无明显规律。免疫共沉淀结果表明：Jun与Fos蛋白存在相互作用，暗示这两个蛋白形成转录因子激活蛋白AP-1，发挥调控作用。用JNK抑制剂SP600125注射到三疣梭子蟹雌蟹后，卵巢组织中jnk基因表达显著下调，酶活性降低，卵母细胞发育受到抑制；Vg基因的表达模式与c-jun/c-fos基因一致，表达量显著下调，且Vn的积累显著减少；表明三疣梭子蟹JNK信号通路受到抑制，导致卵黄积累减少。JNK激活剂Anisomycin注射到三疣梭子蟹雌蟹后，卵巢组织中JNK基因表达显著上调，酶活性升高，表达量显著上调，卵母细胞发育加速，Vn的积累得到显著增加，表明三疣梭子蟹JNK信号通路得到激活，促进卵黄积累。c-jun靶标基因沉默后，卵巢组织中JNK基因的表达量无差异，Vg基因的表达模式与c-jun/c-fos基因一致，表达量显著下调，卵母细胞发育受到抑制，Vn的积累显著减少，表明三疣梭子蟹Jun蛋白参与调控卵黄积累，抑制了卵黄积累。Vg基因沉默后，卵巢组织中JNK基因的表达量无差异，c-jun和c-fos的表达均显著下调，Vg基因的表达量显著上调；但卵母细胞发育受到抑制，Vn积累显著减少，表明干扰Vg基因抑制卵黄积累。综上所述，JNK信号通路通过PtJun-PtFos/PtAP-1转录因子调控PtVg的合成，从而影响三疣梭子蟹雌蟹的卵黄积累。本研究丰富了三疣梭子蟹繁育生物学理论，同时为三疣梭子蟹的养殖提供生产指导。