Nanos2是调控小鼠雄性生殖细胞发育的重要RNA结合蛋白，能促进生殖细胞向雄性分化并维持精原干细胞（spermatogonial stem cells, SSCs）动态平衡，但大家畜Nanos2相关研究缺乏且机制不清楚。本项目拟以水牛为研究对象，主要开展以下研究：①采用Nanos2与候选SSCs膜表面蛋白免疫荧光共染色方法，筛选出水牛SSCs膜特异表面蛋白；②以特异表面蛋白作为标记分选SSCs，构建Nanos2过表达、RNAi和移码突变SSCs细胞系；③观察这三种细胞系的干性特征及表观遗传学变化；④联合分析这三种细胞系TMT外标定量质谱分析数据和转录组测序数据，挖掘潜在参与Nanos2维持SSCs干性特征的重要信号通路。本项目预期结果有助于进一步提高水牛SSCs分选效率，拓展我们对Nanos2维持SSCs干性特征相关分子机制的认知，为推动大家畜SSCs体外培养及分化相关研究奠定理论基础。

Nanos2 is an important RNA-binding protein, which plays a pivotal role in the sexual differentiation of male germ cells and is required for maintaining homeostasis of SSCs. However, the function and the underlying mechanism of Nanos2 in male germ cell of domestic animals are poorly understood, especially in buffalo. This project will focus on the following contents with the buffalo as research object: We firstly investigate co-localization status of NANOS2 and candidate SSCs membrane surface proteins by immunofluorescence staining to screen the specific membrane surface protein of buffalo SSCs. And then SSCs are sorted with the selected biomarker. We establish buffalo SSCs cell lines of Nanos2-overexpresison, Nanos-RNAi and Nanos2-mutation to investigated the changes of stem cell characteristics and epigenetics status in these genetic modified SSCs cell lines. Meanwhile, we jointly analyze the mass spectrometry data collected by high resolution mass spectrometry with TMT labeling and high throughput transcriptase sequence data of these genetic modified SSCs, to further excavate the potential important signaling pathways that participate the function of Nanos2 maintaining the buffalo SSCs characteristic. The expected results of this project will help to improve the efficiency of the buffalo SSCs sorting system and expand our understanding of the molecular mechanisms by which Nanos2 regulates SSC characteristics，laying a theoretical foundation for promoting the related research on the culture and differentiation of domestic animals SSCs in vitro.