凡纳滨对虾新型VIH调控卵黄生成的信号转导机制研究

Pacific white shrimp (Litopenaeus vannamei) is the most important economic culture shrimp species in China. But further development of its aquaculture industry is restricted by the deficiency of current-used methods in artificial-induced ovarian maturation. Vitellogenesis-inhibiting hormone (VIH) is the most effective peptide hormones in regulation of ovarian maturation in crustaceans, via inhibition of vitellogenin (VTG) gene expression. However, studies about the signal transduction mechanism on regulation of vitellogenesis by VIH are still limited. To fill the research gaps in this important scientific question, we first indentified a novel VIH gene in L. vannamei, obtained its purified recombinant protein and examined its inhibitory effects on vitellogenesis via both in vivo and in vitro approaches. We also found that AC/cAMP and NO/cGMP signaling pathways might be involved in regulation of vitellogenesis by VIH. In this project, we are planning to perform the large scale data analysis techniques including transcriptome and proteomics to screen the potential signal pathways in in vitro primary cell culture. We will further study the relative signal pathways by molecular and cellular biological methods including signal pathway activation/blocking and promoter assay. The possible signal pathways involved will be finally examined in in vivo shrimp artificial-induced ovarian maturation experiment. As a whole, our study will describe the signal transduction mechanism of VIH regulation on vitellogenesis at multiple levels. Our study will provide a solid theoretical foundation for developing a novel shrimp artificial-induced ovarian maturation method based on signal pathway activation/blocking.

凡纳滨对虾是我国最重要养殖对虾品种。但当前其雌虾催熟技术存在不足，制约了产业的进一步发展。卵黄蛋白生成抑制激素（VIH）是调控甲壳动物卵巢成熟的关键激素，通过抑制卵黄蛋白原（VTG）的合成起作用，然而相关的信号转导机制研究还非常缺乏。为填补这一重要科学问题的空白，前期我们在凡纳滨对虾中分离了一个新型的VIH基因，获得重组蛋白并验证了它的卵黄生成抑制作用。我们还发现AC/cAMP和NO/cGMP信号通路可能与VIH调控卵黄生成有关。后续研究中，我们拟基于离体细胞培养平台，采用转录组、蛋白质组等大数据分析手段，对VIH调控卵黄生成的信号通路初步筛选；通过信号通路激活/阻断和启动子分析等分子和细胞生物学方法，对涉及信号通路进一步细化研究；并通过活体对虾催熟实验加以验证。本研究从多个方面对VIH调控卵黄生成的信号转导机制进行阐述，将为开发基于信号通路激活/阻断的新型对虾催熟技术提供坚实的理论基础。

凡纳滨对虾是我国最重要养殖对虾品种。但当前其雌虾催熟技术存在不足，制约了产业的进一步发展。卵黄蛋白生成抑制激素（VIH）是调控甲壳动物卵巢成熟的关键激素，通过抑制卵黄蛋白原（Vg）的基因表达和卵黄蛋白（Vn）的合成起作用，然而相关的信号转导机制研究还非常缺乏。为填补这一重要科学问题的空白，前期我们在凡纳滨对虾中分离了一个新型的VIH基因（命名为VIH-2），获得重组蛋白并验证了它的卵黄生成抑制作用。本项目研究发现，肝胰腺是凡纳滨对虾卵巢发育过程中Vg基因表达和Vn合成的主要器官。在肝胰腺中，VIH-2通过非NO依赖的cGMP通路，及下游JNK磷酸化和P38合成，负调控Vg基因表达，而cAMP则是与VIH-2不相干的另一条正调控Vg基因表达的信号途径。基于信号通路研究，本项目还筛选出一种通过环磷酸腺苷类似物和鸟苷环化酶阻断剂促进凡纳滨对虾卵巢发育的方法。此外，本研究发现在凡纳滨对虾中，眼柄窦腺蜕皮抑制激素（MIH）-2而非MIH-1，具有促进肝胰腺卵黄蛋白原（Vg）的基因表达和卵巢发育的作用。而甲壳类心动肽（CCAP）除了能促进心跳外，还能通过刺激蜕皮激素（Ecd）分泌促进卵黄生成。