Amhy决定尼罗罗非鱼性别的分子机制研究

Sex determination has been one of the frontiers in biological research. It is essential for sex control. Efficiency of aquaculture can be greatly improved by culture of mono-sex fish through sex controlled breeding. The Nile tilapia is a worldwide farming fish with the male growing 50% faster than the female. Culture of genetic male tilapia (GMT) is the best way to increase the yields of this important food fish. In addition, tilapia is one of the best animals for studying the molecular mechanisms of sex determination because of the availability of all-female and all-male fish under laboratory condition and the feasibility of genome editing (genetic manipulation). Based on the identification of the sex determining gene Amhy in Nile tilapia, in this project, a series of in vitro and in vivo experiments will be performed to elucidate the genetic basis, signal transduction of Amhy and its relationship with epigenetic modification in sex determination. Genome editing and transgenic over-expression technology will be used to generate the tilapia knockout and transgenic models to analyze the molecular mechanisms of Amhy in sex determination in depth. This project will deep our understanding of fish sex determination and promote sex control in aquaculture. The following aspects will be investigated, 1) the biological effects of SNP in Amhy on sex determination; 2) the signal transduction pathway of Amhy in sex determination; 3) the relationship between Amhy and key genes involved in male and female sex determination; 4) the relationship between epigenetic modification and Amhy in sex determination; 5) the application of Amhy and genome editing in GMT production in aquaculture.

性别决定是生物学的前沿领域之一，性控育种是提高水产养殖效益的重要手段。罗非鱼是世界性养殖鱼类，雄鱼比雌鱼生长快50%，生产和养殖遗传全雄罗非鱼是水产养殖追求的目标。由于在实验条件下可获得全雌、全雄鱼苗，且能够进行基因组编辑，罗非鱼是研究性别决定分子机制的理想模型。本项目在成功图位克隆尼罗罗非鱼性别决定基因Amhy的基础上，拟通过体内、外实验相结合的研究方法阐明其遗传学基础、信号传导途径以及与表观遗传修饰的关联，借助基因组编辑和转基因过表达技术以及雌雄信号通路关键基因敲除模型，解析Amhy决定性别的分子机制，为全面阐明硬骨鱼类性别决定的分子机制奠定基础，并最终服务于水产养殖。研究内容包括：1）单碱基突变对Amhy决定性别功能的影响；2）Amhy的信号传导通路；3）Amhy与雌雄通路关键基因的关系；4）Amhy与表观遗传修饰和性别决定的关系；5）Amhy在两系配套生产遗传全雄罗非鱼的应用。

性别决定是生物学的前沿领域之一，性控育种是提高水产养殖效益的重要手段。本项目在克隆尼罗罗非鱼性别决定基因amhy的基础上，拟阐明其遗传学基础、信号传导途径及与表观遗传修饰的关联，借助雌雄通路关键基因敲除模型，解析amhy决定性别的分子机制。取得以下成果：1）解析了Amhy性别决定的信号传导途径，发现amhy与II型受体amhr2结合招募I型受体alk2/3/6磷酸化Smad1/5/8从而抑制Cyp19a1a的表达和雌激素的合成决定雄性性别，从而揭示了TGF-β信号通路决定鱼类性别的信号传导途径；2）开展了性别决定中amhy新功能化、amhΔ-y去功能化分析，发现amhy成为决定性别不是SNP(C/T)的差异所致，而是由于启动子差异改变表达模式导致的；3）解析了Amhy与雌、雄信号通路关键基因的关系，构建了amhy-dmrt1-gsdf/sox9b-cyp11c1雄性性别决定通路和foxl2-cyp19a1a/E2/esr2a/2b-foxl3雌性性别决定通路；4）阐明了雌、雄性别决定通路关键基因之间的拮抗机制，揭示Dmrt1和Foxl3拮抗性决定鱼类生殖细胞命运的分子机制，提出鱼类生殖细胞的可塑性依赖于dmrt1和foxl3的同时存在的观点。阐明了雌雄通路基因dmrt1、gsdf和foxl2、cyp19a1a之间的拮抗机制，证明dmrt1对雄性精巢发育不可或缺；5）通过基因编辑阐明了类固醇合成酶复制基因在性别决定与分化中的作用差异，发现突变雌激素合成途径的酶基因导致性逆转，而突变雄激素合成途径的酶基因导致精子发生缺陷，丰富了我们对鱼类类固醇合成途径的认识；6）E2/esr可能通过调控DNA甲基化酶，使雄性通路基因的表达抑制而维持雌性表型；7）通过内分泌手段或基因编辑实现罗非鱼的原发和次发性逆转，分析了YY鱼难以诱导性逆转和YY伪雌鱼不育的原因，诱导并培育出可育的YY伪雌鱼，建立了YY维持系，实现罗非鱼的性别控制。在Development、Genetics、Science Bulletin、Endocrinology、Molecular Ecology Resources和水产学报等发表论文40篇。作国际学术会议报告6人次，其中特邀报告2人次，国内会议报告12人次。获重庆市自然科学三等奖1项，申请国家发明专利2项（授权1项）。培养博士后3人，博士生9人，硕士生17人。