特异型Kdm6a在尼罗罗非鱼GSD和TSD中的作用及其机制研究

Nile tilapia males grow faster than females. Temperature-dependent sex determination (TSD) is one type of fish sex determination. Investigating the molecular mechanism of Nile tilapia TSD will be meaningful for enriching fish theory of sex differentiation and all-male Nile tilapia development. Previous transcriptome analysis in our lab revealed that Nile tilapia specific Kdm6a, a demethylase of H3K27me3, could respond to high temperature treatment quickly. Further studies indicated that there was a significant difference in the expression level of specific Kdm6a between males and females in Nile tilapia, and the expression level of specific Kdm6a increased by 20.2 times on the first day after high temperature treatment. Therefore, we hypothesized that specific Kdm6a plays an important role in GSD (Genetic Sex Determination) and TSD in Nile tilapia. To verify this hypothesis, the effects of various treatments on the expression level of specific Kdm6a protein were investigated. Then, CRISPR/Cas9 and transgene technique were used to investigate that specific Kdm6a could affect sex differentiation by regulating the H3K27me3 level in promoter regions of some genes in specific tissues. In the end, RNA-Seq was used to screen differential expression genes between specific Kdm6a overexpressed fish and control fish. ChIP technique determined the regulation role of specific Kdm6a on the H3K27me3 level in promoter regions of screened genes in specific tissues. Collectively, the regulatory mechanism of specific Kdm6a in Nile tilapia GSD and TSD was illuminated.

尼罗罗非鱼雄性生长速度显著快于雌性，研究其性别决定机制，能为全雄尼罗罗非鱼培育提供技术支撑。申请人前期通过转录组分析发现特异型Kdm6a是尼罗罗非鱼响应高温处理的重要基因，它是H3K27me3的去甲基化酶，进一步研究发现特异型Kdm6a在尼罗罗非鱼雌雄鱼间表达水平差异显著，且高温处理后的第1天其表达水平就上调了20.2倍，推测特异型Kdm6a在尼罗罗非鱼遗传性别决定（GSD）和温度性别决定（TSD）调控中均具有重要作用。为了验证该假想，本项目拟进一步分析各种处理对特异型Kdm6a蛋白表达水平的影响；利用基因敲除和过表达等技术研究特异型Kdm6a通过调控特定组织中某些基因启动子区部位的H3K27me3水平，影响尼罗罗非鱼性别分化的分子机理；研究高温通过特异型Kdm6a间接调控的性别分化相关基因及调控作用途径，最终揭示特异型Kdm6a在尼罗罗非鱼GSD和TSD中的作用及其调控机制。

尼罗罗非鱼是世界第二大养殖鱼类，但雄性生长速度显著快于雌性，全雄养殖备受业内相关人士关注。尽管尼罗罗非鱼具有性染色体，是一种遗传性别决定（GSD）的鱼类，但高温也能诱导尼罗罗非鱼雌鱼性逆转为生理型雄鱼，因此，尼罗罗非鱼是一种遗传性别决定（GSD）和温度性别决定（TSD）混合型的鱼类，研究其性别决定的分子机制有助于建立全雄罗非鱼培育的新方法。我们前期通过转录组分析发现特异型Kdm6a是尼罗罗非鱼响应高温处理的重要基因，它是H3K27me3的去甲基化酶，进一步研究发现特异型Kdm6a在尼罗罗非鱼雌雄鱼间表达水平差异显著，且高温处理后的第1天其表达水平就上调了20.2倍，推测特异型Kdm6a在尼罗罗非鱼遗传性别决定（GSD）和温度性别决定（TSD）调控中均具有重要作用。本项目利用制备的抗体揭示了特异型Kdm6a在雄性尼罗罗非鱼性腺中的蛋白表达水平显著高于雌性，高温处理能上调其表达，达到与雄性类似的水平。接着，研究了高温处理对性别未分化的尼罗罗非鱼雌鱼脑和性腺中基因表达的影响；Letrozole处理对尼罗罗非鱼性腺中基因表达的影响，筛选出了除特异型Kdm6a外高温影响的脑和性腺中其他含Jmjc结构域基因，并克隆了含Jmjc结构域基因Jarid2b，研究了高温处理对其表达的特异影响。最后，我们发现，过表达尼罗罗非鱼特异型Kdm6a能显著下调性腺中H3K27me3水平，诱导尼罗罗非鱼雌鱼性逆转，且特异型Kdm6a能通过去除Dmrt1基因启动子区H3K27me3水平上调Dmrt1表达，证明了特异型Kdm6a在尼罗罗非鱼GSD和TSD调控中起重要作用。本研究加深了人们对尼罗罗非鱼性别决定机制的了解，为建立全雄罗非鱼培育的新方法提供了重要基础。