性别相关基因及其甲基化在牙鲆性别表型形成中的内分泌反馈调控研究

The olive flounder, Paralichthys olivaceus, is an important marine economic fish species in China. The culture of all-female flounder has been attracting culturists' interests because the females grow faster and larger than the males. Flounder sex determination bases of genotype by environment interaction. Mounting evidence now suggests that epigenetics is the missing link between genetics, the environment, and endocrine function. However, few papers about the mechanism of epigenetic modification effect on the fish sex differentiation were reported. In this project, at first, we'll produce two stocks of larvae, both of which have XX (genetic females), but each grows up to display all phenotypic females or males, making use of meiotic gynogenetic diploid and sex reversion induction. Then the different expression patterns of genes, such as the brain and gonadal aromatase genes (cyp19) and their transcription factors, steroid receptor genes(estrogen receptor gene and androgen receptor gene), and the epigenetic related genes (dnmt3), during the sexual phenotype formation, will be comparatively studied. We'll also analyze the dynamics of DNA methlytion level in the promoter regions of cyp19s and their transcription factors, and estradiol-17? and testosterone concentrations in larvae or juveniles during this process. Furthermore, the regulation relations among cyp19s and their transcription factors will be studied using EMSA/ChIP and transient transfection methods. Finally, we'll try to reveal the mechanism of epigenetic modification and endocrine feedback regulation of hypothalamus-pituitary-gonad axis (HPG axis) of cyp19s and their transcription factors during flounder sex differentiation. The achievements will promote fish sex control study, establish the theoretical basis for the culture of all female in flatfish, and provide references for mechanism study on fishery resources variation in response to environmental change.

牙鲆是我国重要的海洋经济鱼类，其雌性个体生长快的特性使得全雌养殖倍受重视。牙鲆的性别是由环境和遗传因素共同决定的，表观遗传修饰是这两个因素相互作用进而进行内分泌反馈调控的桥梁，但是表观遗传修饰在鱼类性别分化中作用机制的研究报道还极少。本项目通过染色体操作、分子生物学、内分泌学及表观遗传学等方法，对比分析牙鲆不同性别表型形成过程中，脑型和性腺型芳香化酶基因（cyp19）及其转录因子、性激素受体基因，以及表观遗传相关基因差异表达谱；同时对性别相关基因启动子区DNA甲基化水平和鱼体内性激素水平变化规律进行明确，并研究cyp19及其转录因子之间的调控关系，以期阐明该过程中这些基因的表观遗传修饰及下丘脑-垂体-性腺轴的内分泌反馈调节作用机制。获得的成果既可以跟踪国际前沿，对鱼类性别控制的研究带来促进作用，也为牙鲆等鲆鲽鱼单性群体养殖提供理论依据，并为渔业资源响应环境变化的变动机制研究提供参考。

该项目以我国重要海水养殖鱼类牙鲆为对象，对鱼类性腺分化中起主要作用的雌二醇合成关键酶基因cyp19进行研究，不仅对比分析该基因及其重要转录因子在性别表型形成过程中的差异表达，探讨表达水平与基因启动子区DNA甲基化水平，以及与体内性激素水平的关联性；而且初步研究了这些基因之间的调控关系。首先，从卵巢、精巢转录组数据中分别筛选到2,193和887个差异表达基因，预测了多个与性别相关的通路，包括雌激素合成通路；结合同源克隆等，获得nr5a2、nr0b1、cyp19a、ar、wnt4等多个基因序列，并经验证均为性别相关基因；定量PCR分析发现，这些基因分别参与牙鲆原始性腺、精巢或卵巢分化的过程。同时，进行了表观遗传学研究，主要包括牙鲆DNA从头甲基化转移酶基因dnmt3的克隆和差异表达分析，以及上述基因启动子区DNA甲基化水平变化。分析表明dnmt3a和dnmt3b参与了性别分化过程；在该过程中，性类固醇激素、温度等外界因子可以影响到cyp19a等基因启动子区的甲基化水平，可能导致了基因两性差异表达和雌雄性腺的形成。对牙鲆性别表型形成过程中雌雄类固醇激素水平的变化规律也进行了跟踪监测, 发现无论温度还是激素处理，都会影响体内性激素水平，由此改变牙鲆性别分化的方向。进一步，探讨了cyp19a基因表达的调控，发现Ar、Dmrt1、Nr5a2及Nr0b1均对cyp19a启动子具有作用，Nr5a2和Nr0b1在cyp19a调控上存在拮抗关系；cyp19a启动子是受其甲基化水平调控。综上所述，cyp19a在牙鲆性别分化中起重要作用，外源雌二醇诱导雌核发育幼鱼向着卵巢形成方向分化，而高温诱导引起cyp19a启动子区高甲基化水平和转录因子nr0b1、dmrt1的上调表达，导致cyp19a转录水平降低，雌二醇合成水平受到限制，其合成前体睾酮的水平升高，向11-酮基睾酮合成方向转化，从而促进了精巢形成方向的分化。. 本项目从分子生物学、细胞生物学、表观遗传学、内分泌学等水平阐述了cyp19及其重要转录因子表观遗传修饰及内分泌反馈调节在牙鲆性别表型形成中的作用。相关研究工作有些在牙鲆中甚至鱼类中首次开展，将有助于鱼类性腺分化机制的解析。