三疣梭子蟹胰岛素样促雄性腺激素基因功能及其调控研究

The mechanism of sex determination and differentiation in decapods is a hot research topic for crustacean developmental biology. The hologynic culture of the swimming crab Portunus trituberculatus is provided with great economic values. It is well known that the insulin-like androgenic gland hormone (IAG) gene plays a crucial roles on male sex differentiation and sexual characteristics development of decapods. Based on a well understanding of the histology of androgenic gland of P. trituberculatus, and the preliminary result of the cDNA cloning and tissue distribution of IAG mRNA, this project aims to illustrate the regulatory roles of IAG gene on gonadal development, gametogenesis, secondary sexual characteristics development, molting and growth, and to confirm the time window of IAG gene in controlling the male sexual differentiation of P. trituberculatus, with the integrated utilization of RNA interference, in vivo injection of recombinant protein of IAG, and in vitro incubation. In addition, the project will aim to identify the 5’-flanking sequence of IAG gene, analyze its transscription factor binding sites and the promoter transcriptional activity, and to reveal the mechanism of transcription factors, miRNAs, neurotransmitters, and CHH family neuropeptides in regulation of IAG gene expression. To carry out this research will provide theoretical guidance for sex reversion and all-female population culture of P. trituberculatus, and help to elucidate the sex determination and differentiation mechanism of this species.

虾蟹类性别决定和性分化机制是甲壳动物发育生物学研究的一个热点。三疣梭子蟹单雌化养殖能显著提升其人工养殖的经济效益。胰岛素样促雄性腺激素（IAG）基因被认为在虾蟹类雄性分化和雄性性征的发育上起关键的调控作用。在已经掌握三疣梭子蟹促雄性腺组织学特征、克隆IAG全长cDNA序列并分析其组织差异表达的基础上，拟进一步综合应用RNAi技术、活体注射重组蛋白（rIAG）和离体培养等方法阐明IAG基因对性腺发育、配子发生、第二性征发育、蜕皮和生长等生理过程的调控作用，确定IAG基因调控三疣梭子蟹雄性分化的时间窗口；克隆分析IAG基因启动子和表达调控序列，揭示转录因子、miRNAs、神经递质和CHH家族神经肽对IAG基因表达的调控机理。开展本项研究，将为实现三疣梭子蟹性反转和单雌化养殖提供理论指导，还将有助于阐明其性别决定和分化机制。

胰岛素样促雄性腺激素（IAG）被认为在虾蟹类雄性分化和精巢发育上起关键的调控作用。为阐明三疣梭子蟹IAG基因（PtIAG）功能，采用qPCR技术检测了PtIAG在个体发生和精巢发育过程中的表达水平变化，发现PtIAG与精巢发育和精子发生密切相关；利用原核表达载体pET-28a-sumo和大肠杆菌Rosetta（DE3）菌株实现了rIAG的原核表达，并对重组蛋白进行了质谱鉴定。为探讨IAG基因作用的分子机理，克隆获得IGFBP基因和2条IR基因，PtIR主要表达于促雄腺与发育的精巢；通过共转染实验和Western blot检测，发现PtIR与PtIAG共定位，PtIR与IAG结合后通过磷酸化级联放大反应激活胰岛素通路下游的MAPK蛋白，进而调控精巢发育。为揭示IAG基因表达的调控机制，克隆获得PtIAG基因的5’上游调控序列；构建含有启动子区的报告载体pEGFP-1-PtIAG将其转染至HEK293T细胞系中，结果显示pEGFP-1-PtIAG能驱动EGFP蛋白的表达；构建不同长度的启动子-双荧光素酶报告基因载体，将它们分别转染至HEK293T细胞系中，通过活性比较确定在该启动子序列的+152至-941之间存在核心启动子区；鉴定出三疣梭子蟹47条神经肽及其受体，分析了神经肽及其受体转录本在不同组织的分布情况，为进一步研究神经肽对IAG基因表达的调控奠定了基础；通过RNAi和离体培养实验，发现PtCHH、PtCFSH对PtIAG起正向调控作用，而PtMIH则对PtIAG存在抑制性调控作用；通过离体共培养和活体注射实验，发现5HT与DA对精巢发育具有相互拮抗的调控作用。这些研究成果不仅有助于揭示三疣梭子蟹性别分化和精巢发育机理，而且能为三疣梭子蟹性控技术的建立提供理论支持。