The sex determination and control of poultry is a key problem in production and scientific research despite few studies on regulatory factors, especially transcription factors in sex determination. In the early stage of this study, high-throughput sequencing was used to screen the differentially expressed gene JUN in male and female embryonic stem cells (ESCs) and primordial germ cells (PGCs). The qRT-PCR discovered that the JUN gene significantly increased from embryonic days (E) 2.5 later in chicken embryo development, and the female gonad expression was much higher than that of the male after E14.5. Lentivirus shRNA-JUN, shRNA-Smad2 interference, and OE-JUN overexpression vectors were successfully constructed. After interfering with JUN in vivo, male characteristics appeared in ZW embryonic gonads at E18.5. Meanwhile, the male-specific genes DMRT1 and Sox9 were upregulated, the female-specific genes FOXL2, ESR1, and CYP19A1 were downregulated, and the estradiol in the gonads was significantly decreased. The situation was reversed after the overexpression of JUN, ZZ chicken embryo developed into female sexual characteristics. The double luciferase report has found that the Smad2 promoter activity was significantly upregulated after interference with JUN, and significantly increased after the deletion of the JUN binding site. After the injection of the Smad2-shRNA vector into the blood vessel in vivo, it was discovered that DMRT1 and Sox9 of ZW embryos at E18.5 were downregulated, FOXL2 and CYP19A1 were significantly upregulated, and the gonads show femininity. In conclusion, this study proves that JUN is a key regulator in the process of chicken female sex differentiation, which can inhibit the transcription of Smad2 and promote the synthesis of estradiol, and participate in the process of chicken sex differentiation. This study lays a foundation for the analysis of the molecular mechanism of chicken sex determination and the development of poultry sex control technology.

家禽的性别决定和控制是生产和科学研究中的一个关键问题，尽管对调控因素特别是性别决定中的转录因子的研究很少。本研究前期采用高通量测序技术筛选男女胚胎干细胞（ESCs）和原始生殖细胞（PGCs）中差异表达基因JUN 。qRT-PCR发现JUN基因在鸡胚发育后期从胚胎天数（E）2.5开始显着增加，E14.5后雌性性腺表达量远高于雄性。慢病毒 shRNA -JUN、 shRNA- Smad2干扰和OE- JUN成功构建过表达载体。在体内干扰JUN后，ZW胚胎性腺在E18.5出现雄性特征。同时，雄性特异性基因DMRT1和Sox9上调，雌性特异性基因FOXL2、ESR1和CYP19A1下调，性腺中雌二醇显着降低。过表达JUN后情况发生逆转，ZZ鸡胚发育成雌性性征。双荧光素酶报道发现，干扰JUN后Smad2启动子活性显着上调, 并在删除JUN结合位点后显着增加。在体内将Smad2 -shRNA载体注入血管后，发现E18.5时ZW胚胎的DMRT1和Sox9下调，FOXL2和CYP19A1显着上调，性腺表现出女性化。综上所述，本研究证明JUN是鸡雌性分化过程中的关键调控因子，可抑制Smad2的转录。促进雌二醇的合成，参与鸡的性别分化过程。本研究为分析鸡性别决定的分子机制和发展家禽性别控制技术奠定了基础。