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。

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.