miRNA/novel_167靶向抑制Dmrt1的表达在红鳍东方鲀性别分化过程中的功能研究

Accumulating evidence suggests that miRNAs play important roles in sex differentiation of animals, however, the regulatory and functional roles of miRNAs in gonadal sex differentiation have not been well clarified in fish yet. The tiger pufferfish (Takifugu rubripes) was considered as one of the most economically important marine fish in China. As male fugu are more valuable than female, study on the molecular mechanism of sex determination and differentiation of this species is essential for achieving all-male mono-sex culture. Dmrt1 has a conserve role in the signaling pathway during male sex-determination sex-differentiation across vertebrates. Our previous studies systematically analyzed the miRNAs expression profiles in undifferentiated gonad tissues and screened numerous sex-bias expressed miRNAs in Takifugu rubripes. Among them, miRNA/novel_167 were expressed higher in female gonads and predicted to target Dmrt1. Therefore, in this project, we will use qPCR, in situ hybridization and dual-luciferase reporter assay to investigate whether miRNA/novel_167 negatively regulate the Dmrt1 expression firstly. Then, miRNA/novel_167 knockdown and overexpression experiments will be carried out to figure out the functions of miRNA/novel_167 in sex differentiation. Finally, gene knockdown and hormone treatment will be performed to study the relationship of miRNA/novel_167 with key genes involved in male and female sex determination. The results will help us to clarify the role of miRNAs in sex determination and differentiation in fish.

miRNAs在动物的性别分化过程具有重要作用，但其在鱼类性别分化过程中的功能和作用机制尚不明确。红鳍东方鲀是我国重要海水养殖经济鱼类，雄性商品鱼价格较高，对其性别决定和分化机制的研究是生产上实现单雄养殖的必要基础。Dmrt1基因是脊椎动物雄性性别决定和分化通路上的保守基因。我们前期在未分化性腺中筛选了性别差异表达的miRNAs，其中，miRNA/novel_167在雌性性腺高表达，且靶基因是Dmrt1。本项目拟在前期工作的基础上，首先采用qPCR、原位杂交、双荧光素酶报告基因等技术进一步明确miRNA/novel_167对Dmrt1的靶向抑制关系。然后，通过miRNA/novel_167敲降和过表达实验研究其在性别分化过程中的功能。最后通过基因敲降和激素处理实验，研究miRNA/novel_167与雌、雄通路关键基因之间关系。研究结果将为解读miRNAs在鱼类的性别分化过程的功能提供参考。

红鳍东方鲀是我国重要海水养殖经济鱼类，雄性商品鱼价格较高，对其性别决定和分化机制的研究是生产上实现全雄养殖的必要基础。近年来，越来越多的研究发现非编码RNAs在动物的性别分化过程具有重要作用，本研究首先利用高通量测序技术对河鲀幼鱼未分化性腺进行全转录组测序分析，筛选了性别差异表达的miRNA、lncRNA和cirRNA，其中，与XX性腺相比，分别有3个成熟 miRNAs, 79个lncRNAs和34个cirRNAs在XY性腺中上调，5个成熟miRNAs, 51个lncRNAs和26个cirRNAs下调。在这些差异表达的非编码RNA中，miRNA/novel_167在雌性性腺高表达，且预测靶基因是雄性性别分化相关基因Dmrt1。采用双荧光素酶报告基因进一步明确miRNA/novel_167对Dmrt1的靶向抑制关系。然后，对河鲀幼鱼进行AI、MT或E2处理，结果发现，AI和MT可诱导XX河鲀雄性化，E2可以诱导XY河鲀雌性化。转录组分析后发现在性别转变过程中dmrt1, gsdf, foxl2以及其它与类固醇激素合成相关的基因 (如star, hsd3b1, cyp11c1, cyp19a1a, etc.) 表达发生变化。此外，转录组和qPCR结果均显示在对照组XY性腺中，dmrt1和gsdf的表达显著高于XX性腺；在对照组XX性腺中，foxl2和cyp19a1a的表达显著高于XY性腺；AI处理后，XX性腺中的foxl2和cyp19a1a的表达水平降低，dmrt1和gsdf 表达上升；MT处理后，XX性腺中的foxl2, cyp19a1a, dmrt1和gsdf的表达均下降；E2 处理后，XY性腺中dmrt1和gsdf的表达水平下降。因此，上述基因在河鲀的性别分化过程中具有重要作用。同时，还发现，AI处理后，XX性腺中novel_167的表达水平下降，而E2处理后，XY性腺中novel_167的表达水平上升。最后，novel 167 agomir和antagomir性腺注射实验表明novel 167可在体下调dmrt1基因的表达水平，但敲降dmrt1对novel 167的表达无影响。研究结果将帮助我们解析miRNAs和其他非编码RNA在河鲀的性别分化过程的功能。