随着全球气候变暖，热应激已成为奶公牛精液品质下降的重要原因。Hsp72在抵抗热应激致细胞的氧化损伤中发挥重要作用，但具体分子机制不明。本项目拟以Hsp72抵抗热应激致奶牛睾丸支持细胞的氧化损伤为假说，以Nrf2/Keap1/ARE信号通路在热应激条件下的反应变化为研究切入点。利用CRISPR/Cas9技术建立Hsp72敲除细胞模型，检测热应激对细胞氧化还原的影响，明确Hsp72抵抗热应激致细胞氧化损伤的作用；采用IP-MS及RNA-seq等分子生物学手段，探究热应激条件下Hsp72对Nrf2复合体组成、核转位及其下游基因表达的影响，阐明Hsp72调控Nrf2信号通路活化的分子机制；通过功能拯救实验，分析Hsp72敲除细胞中Nrf2激活剂对热应激致细胞氧化损伤的影响，揭示Hsp72调控Nrf2信号通路抵抗热应激致细胞氧化损伤的关系网络；为提高夏季奶公牛的精液品质和繁殖力提供理论和实验依据。

With the global warming, heat stress has become an important reason for the decrease of dairy bull semen quality. As an important heat shock protein (Hsp), Hsp72 plays an important role in resisting oxidative damage caused by heat stress, however, its specific molecular mechanism underneath is unknown. In this project, we propose that 'Hsp72 can resist oxidative damage caused by heat stress in sertoli cells of dairy bull' as a hypothesis, and take the changes of Nrf2/Keap1/ARE signaling pathway under heat stress as an entry point. The model of Hsp72 knockout cells will be established by using CRISPR/Cas9, and used to detect the effect of Hsp72 on REDOX of the cells under heat stress, in order to clarify the role of Hsp72 in resisting oxidative damage caused by heat stress. IP-MS and RNA-seq will be used to explore the effects of Hsp72 on Nrf2 complex formation, nuclear translocation and its both up- and down-stream genes expression under heat stress for the purpose of clarifying the molecular mechanism of Hsp72 in regulating the activating Nrf2 signaling pathway. Finally, the effect of Nrf2 activator on oxidative damage caused by heat stress will be analyzed in the Hsp72 knockout sertoli cells of dairy bull through functional rescue experiment in the aim of revealing the network of Hsp72 in regulating Nrf2 signaling pathway to resist oxidative damage caused by heat stress. The results of the study will provide a theoretical and experimental basis for improving semen quality and fecundity of dairy bull in summer.