牙鲆是我国重要的海水养殖对象，雌性比雄性生长快，理解其性腺分化与发育的遗传机制是有效进行性别控制的前提。cbx2基因通过调节SF1、FOXL2等基因表达可影响哺乳动物性腺分化的方向，其基因突变或功能缺失会导致性反转。我们在牙鲆中意外发现cbx2是众多雌雄性腺差异表达miRNA的潜在靶基因，siRNA实验初步表明多个性别相关基因（sf1、sox9、foxl2、wnt4）表达明显受到cbx2表达水平的调节，双荧光素酶报告实验也初步证实cbx2是上述雌雄性腺显著差异表达的miR-200家族直接作用的靶基因，提示该基因在miRNA调节牙鲆性腺分化与发育的分子网络中可能发挥着关键介导作用。因此，本项目拟聚焦cbx2和miR-200家族基因，将miRNA与特定基因表达联系起来，通过基因表达谱分析、细胞与活体水平的基因过表达或抑制等多层面解析cbx2介导miR-200家族在牙鲆性腺分化与发育中的功能。

The Japanese flounder (Paralichthys olivaceus) is an important commercially cultured marine flatfish in China, of which female grows faster than male. And thus investigation on the molecular regulatory mechanism of gonad differentiation and development in this species is of great significance in aquaculture. CBX2, a member of the PcG family, is an important transcription factor and can mediate the gene expression of nr5a1/SF1 or foxl2, and therefore plays a key role in the sex development of mammals. Genetic mutations or lack of function of the CBX2 can lead to sex reversal in mammals. However, little is known of the molecular mechanisms of cbx2-regulated gonad differentiation and development in fish. In our previous study on gonad-related miRNA screening and function analysis in the flounder, it was found that cbx2 is a potential target gene for many sex-biased miRNAs. Our results from cbx2 siRNA experiment have shown that the gene expressions of several sex-related genes, such as sf1, sox9, foxl2 and wnt4, are regulated by the cbx2 gene. Dual-luciferase reporter assay preliminary has demonstrated that the cbx2 is a target gene of miR-200 family genes (miR-200a, miR-200b, miR-141 and miR-429), which exhibit obvious differential expression in hermaphroditic glands. For this reason, the cbx2 may act as an extremely important node to mediate miRNA/sex-related genes regulatory network. The project's objective is to focus on the cbx2 and miR-200 family genes to investigate the expression patterns by fluorescent quantitation PCR, in situ hybridization and immunohistochemistry, and analyze the gene over-expression, knockdown or knockout in vivo and in vitro by RNAi, antisense RNA and miRNA mimics or antagomir experiments. And thus further clarify to the molecular mechanism of cbx2-mediated miR-200 family regulation in gonad differentiation and development of Paralichthys olivaceus.