黄河鲤早期性腺分化发育关键期的确定及其基因表达谱分析

It is still unclear for the mechanism of gonad development and differentiation in vertebrate until now, however, due to the original of gonadal development and the diversity of the sex decisions in fish, which provided a good experimental materials to study on the mechanisms of gonadal differentiation and development in vertebrates. The Yellow River carps were valuable fish resources and had important economic value. The project intended to distinguish the key period of the early stage of sperm or ovarian differentiation by the methods of histological and immunohistochemical in the Yellow River carp; and the Laser capture microdissection was used to separate the gonads before and after gonadal differentiation for analyzing the gene expression profiles of each period of the gonad development by zebrafish genome chip as a template, which depicted the network regulation mechanisms of early gonadal differentiation in the Yellow River carp. In addition, we again selected some testis differentiation associated genes (dmrt1, and sox9, wt1a obtained early) and ovarian differentiation genes (ZP3C and CYP19) to analyze their expression changes before and after gonadal differentiation by quantitative PCR and situ hybridization in the Yellow River carp. We submit the project proposals to lay the foundation for explore the possible molecular and physiological mechanisms of gonadal development of the Yellow River carp and other vertebrates, as well as provide theoretical guidance for artificial control the gender and scale of breeding of the Yellow River carp.

脊椎动物性腺发育与分化机制目前仍不清楚。而鱼类由于其性腺发育的原始性及性别决定类型的多样性，为研究脊椎动物性腺分化发育机制提供了良好的材料；黄河鲤是宝贵的鱼类资源，具有重要的经济价值；本项目拟通过性腺分化的组织学及免疫组化研究，确定黄河鲤早期精、卵巢分化的几个关键时期；应用激光捕获显微切割技术分离黄河鲤性腺分化中各关键期的性腺组织，以斑马鱼基因组芯片为模板，分析黄河鲤各时期的基因表达谱，从而描绘出黄河鲤早期性腺分化的网络调控机制；同时结合精巢分化相关基因dmrt1及前期获得的sox9、wt1a基因等，以及卵巢分化相关基因ZP3C和CYP19等基因，应用荧光定量PCR和原位杂交等技术分析黄河鲤性腺分化过程中相关功能基因的表达变化及组织定位。研究成果将有助于阐明黄河鲤及其它脊椎动物性腺分化的分子机制，同时将为黄河鲤性别的人工控制和规模化繁育提供理论指导。

针对鱼类性腺发育与分化机制目前仍不清楚的问题，本研究通过详尽的组织学与分子生物学手段探寻黄河鲤早期性腺分化发育的机制。主要开展了如下几方面的研究：1）通过性腺分化的组织学及免疫组化研究，确定出黄河鲤早期精、卵巢分化的几个关键时期；2）应用显微切割技术对不同发育期的雌、雄性腺组织进行了分离并纯化其mRNA，分别成功构建了雌、雄未分化时期、性腺分化Ⅱ期（刚分化时期）、Ⅳ期（性腺成熟期）转录组文库，通过对其文库进行深度挖掘，初步了解了雌、雄性腺发育中基因网络调控机制，同时也为将来进一步探索该物种的性腺发育机制提供了大量的基因鉴定资源；3）通过对部分可能参与性腺发育的差异基因（wnt4，dax1，fox12）进行了全长克隆和功能分析，初步阐明这些基因在性腺发育中的重要作用；4）从 mRNA 水平上，分别以未分化性腺与二期卵巢为验证方，以成熟卵巢为检测方，成功构建了黄河鲤未分化性腺和二期卵巢的SSH文库，首次发现bmp家族基因（bmp1b、bmp2b、bmp3、bmp4、bmp5、bmp6、bmp8、bmp15、bmpr1aa、bmpr1ab、bmpr1ba、bmpr1bb、bmpr2a、bmpr2b）在黄河鲤成体和幼体雌雄性腺以及未分化性腺有重要的调控作用。项目研究结果无疑将有助于阐明黄河鲤及其它脊椎动物性腺分化的分子机制。