高温诱导尼罗罗非鱼雌鱼性逆转的分子表观机制

The Nile tilapia is a species of tilapia, a cichlid fish native to Africa. China is by far the main tilapia producing country, with more than 1 million tonnes production each year. The Nile tilapia shows sexual growth dimorphism and the males grows faster than females. As a result, the development of an all-male Nile tilapia stock would be of significant benefit. Nowadays, we all know that high-temperature can induce the female sex reversal. High-temperature induction could be used as a consumer- and environment-friendly approach to significantly increase the proportion of males in Nile tilapia. However, the molecular and epigenetic mechanisms of high-temperatue inducing sex reversal of Nile tilapia females remains elusive. In this study, the differential expression genes between high-temperature induction and control group will be screeened based on transcriptome and methylome analysis. The cellular localization of selected genes will be effectively demonstrated by in-situ hybridiztion and immunohistological analysis. The protein expression changes of seleceted genes after high-temperature induction will also be analyzed. Based on the the result of protein analysis, 1-2 target genes will be selected and used for further studies. In order to analyze the mechanism behind the expression manipulation of target genes, the changes of methylation level of target genes under high-temperature masculinization will be studied. The binding of transcription factor to DNA promoter regions upstream of gene transcription start sites (TSSs) is one of the most important mechanisms by which gene expression, and thus many cellular processes, are controlled. In this study, we will use ChIP for the analysis of transcription factor binding to the promoter of target genes. In the end, Cas 9 will be used for the editing of target genes. We will investigate the the sex reversal rate of knockdown or knockout individuals under high-temperature masculinization. Collectively, this study is meant to provide theoretical and technical foundation for all-male Nile tialpia stock,and improve the understanding of temperature-dependent sex determination in fish.

罗非鱼雄性生长速度显著快于雌性，一直以来人们都致力于培育高雄/全雄罗非鱼。利用高温诱导下罗非鱼雌鱼能性逆转为生理雄鱼的特点，可以建立一种经济环保的培育高雄/全雄罗非鱼的新方法。但是目前我们对高温诱导罗非鱼雌鱼性逆转的分子表观机制仍认识不够。本项目首先基于组学分析找出高温胁迫下调控罗非鱼雌鱼性逆转的候选基因；根据筛选基因的表达定位等情况选择1-2个基因作为目标基因进一步研究；从高温胁迫下目标基因启动子（顺式作用元件）表观遗传修饰改变和转录因子（反式作用因子）与目标基因启动子结合能力变化2个方面揭示高温诱导调控目标基因表达的分子机理；进一步利用CRISPR/Cas 9技术敲降/敲除目标基因，研究高温对敲降/敲除个体性逆转的影响，从基因功能角度探讨高温胁迫下目标基因调控罗非鱼性逆转的分子机制。本项目不仅为培育高雄/全雄罗非鱼提供理论和技术支持，也有助于揭示温度影响鱼类性别分化的分子机理。

尼罗罗非鱼雄性生长速度显著快于雌性，一直以来人们都致力于培育高雄/全雄尼罗罗非鱼。利用高温能诱导尼罗罗非鱼雌鱼能性逆转为生理雄鱼的特点，可以建立一种经济环保的培育高雄/全雄尼罗罗非鱼的新方法。但是目前我们对高温诱导尼罗罗非鱼雌鱼性逆转的分子表观机制仍认识不够。本项目利用转录组和MeDIP甲基化组分析技术研究高温处理在mRNA表达水平和DNA甲基化水平影响的性别分化相关基因，发现高温处理能显著下调Cyp19a1a等雌性相关基因的表达水平，显著上调了Dmrt1、GSDF等雄性基因的表达水平；接着重点以Cyp19a1a为目标基因进行了相关研究，发现高温处理能够显著升高雌雄尼罗罗非鱼Cyp19a1a启动子区的甲基化水平，降低其表达量；染色质免疫共沉淀（ChIP）检测显示，高温诱导能显著升高雄性尼罗罗非鱼组蛋白H3K9的甲基化水平，并且募集到的RNA聚合酶的量显著下降；测定了高温处理组和不同浓度Letrozole处理组的E2、11-KT含量、芳香化酶活性、各处理组的雄性率，发现高温处理组E2含量显著下调，E2水平与1.5 ug/L的letrozole处理组（XX+L1.5）基本一致，但XX+L1.5的雄性率是59.38%，XX+HT组的雄性率却是68.75%，因此高温下调E2通路是高温诱导尼罗罗非鱼雌鱼性逆转的重要调控通路，但还存在其他调控通路参与调控了尼罗罗非鱼雌鱼性逆转。E2补偿策略显示，21dpf时，XX组（2155.27±376.62pg/g）和XX+HT+E:4组（1993.81±133.63 pg/g）的E2水平非常接近，有趣的是，XX组的雌性率（76.36%）和XX+HT+E:4组的雌性率（80.36%）也非常接近。结合以前的结果可以说明E2的下调是导致尼罗罗非鱼雌鱼在性别分化关键期发生性逆转的主要原因。免疫组化分析揭示了在发育早期高温处理的尼罗罗非鱼雌鱼在基因表达水平、表达位置与细胞形态上与雌鱼对照相似，在后期发育的与雄性对照相似。最后，我们发现高温处理能影响脑中Kdm6a、Jarid2等组蛋白甲基化调控相关基因的表达水平，影响体内的H3K27me3水平，将组蛋白甲基化-DNA甲基化-性别相关基因表达关联到一起。本项目揭示了一条高温诱导尼罗罗非鱼雌鱼性逆转的重要的分子通路，为高雄/全雄罗非鱼培育提供了重要的基础。