半滑舌鳎性逆转跨代遗传的表观调控机制

Epigenetics, involving the interaction between genomic and environmental information, is considered the second coding for life forms. Fish sex determination and differentiation are processes where to investigate the influence of epigenetics versus classical genetics. In a previous study, we found the existence of transgenerational inheritance of sex reversal in tongue sole, leading to a non-adaptive sex ratio that is skewed towards predominantly male offspring, thus affecting the development of large-scale tongue sole industry. In this study, taking such an intriguing phenomenon as a starting point, we plan to carry out the whole-genome methylation sequencing and transcriptome sequencing for the early development stages of tongue sole pseudo-male (ZW♂) and normal male (ZZ♂) offspring, respectively. First, we will create dynamic maps of methylation patterns and gene expression profiles related to different aspects of sex determination. Further, we will screen the specific genes that are expressed in each developmental stage and those genes that experience a shift of expression between different stages, revealing the dosage expression mechanism of Z-linked genes after the inactivation of W sperm in pseudo-male (ZW♂). Meanwhile, we will identify the differentially methylated genes (DMGs) between pseudo-male (ZW♂) and normal male (ZZ♂) on the corresponding stages of early development of the offspring. Consequently, we can isolate the imprinted genes involved in the transgenerational inheritance of sex reversal in tongue sole. We will then silence the imprinted genes by RNAi to elucidate the molecular mechanism of imprinted genes that escape genomic reprogramming in the development of pseudo-male offspring, thereby revealing the transgenerational epigenetic inheritance of sex reversal in half-smooth tongue sole mediated by monoallelic expression of Z chromosomal genes.

表观遗传介导基因和环境互作，被喻为生命天书的第二种编码形式。鱼类性别决定和分化是理解基因和环境相互博弈的绝佳案例。本项目组前期研究发现半滑舌鳎存在环境介导的性逆转跨代遗传现象，导致实际生产中雌雄比例严重失调，从而影响半滑舌鳎产业的规模化发展。因此，本项目拟以半滑舌鳎性逆转跨代遗传为出发点，采用高通量测序技术开展半滑舌鳎伪雄鱼（ZW♂）和正常雄鱼（ZZ♂）后代早期发育的全基因组甲基化测序和转录组测序，绘制早期发育的基因动态表达图谱和甲基化图谱。筛选表征伪雄鱼后代早期发育各阶段的特异基因，揭示伪雄鱼W精子失活后Z染色体基因的剂量平衡机制。同时鉴定伪雄鱼（ZW♂）和正常雄鱼（ZZ♂）后代早期发育各阶段的差异甲基化基因，筛选参与跨代遗传的印记基因。利用RNAi技术特异性沉默关键印记基因，阐明印记基因在伪雄鱼后代基因组重编程中逃逸的分子机制，从而解析半滑舌鳎Z染色体介导的性逆转跨代显性遗传机制。

半滑舌鳎是我国北方沿海重要的养殖鱼类，其雌雄个体生长差异显著，雌鱼大小可达雄鱼的2-4倍。人工养殖群体中雄鱼比例高达70%，而雌鱼比例不到30%，雌雄比例严重失衡成为阻碍半滑舌鳎产业规模化发展的瓶颈。前期研究发现半滑舌鳎养殖群体中存在性逆转现象，并且这种性逆转可跨代遗传，导致实际生产中雌雄比例严重失调。因此，本项目拟以半滑舌鳎性逆转跨代遗传为出发点，从表观遗传角度分析半滑舌鳎伪雄鱼和正常雄鱼后代早期发育过程的甲基化变化规律，阐明半滑舌鳎性逆转的跨代遗传机制。本研究构建了6组半滑舌鳎正常雄鱼和伪雄鱼半同胞家系，利用半同胞家系材料，完成了伪雄鱼和正常雄鱼家系早期胚胎发育阶段的转录组测序和甲基化测序，分析了各样本之间差异表达基因和差异甲基化区域，绘制了半滑舌鳎早期发育时期的动态表达图谱和全基因组甲基化图谱。结果表明精子甲基化水平最高，卵母细胞最低，甲基化水平随着早期胚胎发育阶段的变化而逐渐升高，1k细胞时期、囊胚期和原肠期的甲基化水平接近精子的水平。随着胚胎发育的进程，卵细胞特异性甲基化CG位点动态变化规律表明由卵细胞提供的甲基化模版正逐渐被精细胞的模版所取代。进一步分析了伪雄鱼和正常雄鱼精子之间的差异甲基化区域及其在不同染色体之间的分布规律，发现伪雄鱼和正常雄鱼精子之间的差异甲基化类型主要为CpG，并且两者之间的差异甲基化区域显著富集于Z染色体上，同时差异甲基化区域被家系后代较好的继承下去，揭示了半滑舌鳎性逆转的跨代遗传机制。利用体外甲基化修饰法分析了性别决定基因dmrt1差异甲基化区域启动子的转录活性，结果表明与非甲基化启动子比较，差异甲基化区域启动子转录活性均显著下降。此外，分析了性别相关基因patched1、β-catenin1等的表达特征及甲基化模式。上述研究揭示了半滑舌鳎Z染色体连锁的dmrt1基因介导的性逆转跨代遗传机制，为半滑舌鳎高雌苗种制种技术的建立提供了理论依据和技术支撑。