泛素连接酶Neurl3参与半滑舌鳎精子发生的调控机制研究

半滑舌鳎伪雄鱼精子发生异常是导致养殖群体高雄化的主要因素，明确精子发生关键基因的功能及作用机制，对实现性控育种具有重要意义。前期研究发现，泛素连接酶基因neurl3通过泛素化途径参与半滑舌鳎精子发生，其在雄鱼和伪雄鱼中的表达剂量差异与泛素化调控密切相关，然而调控机制尚不明确。据此，本项目拟以neurl3基因作为研究对象，开展如下研究：（1）检测neurl3在雄鱼和伪雄鱼精子发生中的时空转录和翻译水平以及精巢细胞定位，确定其发挥作用的关键时期和细胞类型；（2）运用RNA干扰、基因编辑和过表达技术实现neurl3的敲降、敲除和过表达，进而通过表型检测研究neurl3在精子发生中的功能；（3）利用免疫共沉淀和体外泛素化等技术筛选Neurl3蛋白的泛素化底物，结合RNA-seq和共表达分析构建调控网络，比较neurl3在雄鱼和伪雄鱼精子发生中调控机制的差异，为开展半滑舌鳎性控研究提供理论基础。

In Chinese tongue sole, the abnormal spermatogenesis in pseudomale fish is the predominant factor leading to the high male ratio of culturing population. Therefore, to elucidate the function and regulatory mechanisms of the pivotal genes in spermatogenesis is particularly important for the high productivity in cultivation industry. Our previous study has found that the ubiquitin ligase gene neurl3 is involved in spermatogenesis via ubiquitination pathway, and the dosage discrepancy between male and pseudomale is closely associated with the differential regulation. However, the detailed regulatory mechanism of neurl3 in spermatogenesis remains elusive. Based on these data, in this project we will further focus on the neurl3 gene and plan to initiate the following experiments: (1) We will examine the spatiotemporal transcription and translation levels of neurl3 and clarify its mRNA and protein localization in testicular cells during male and pseudomale spermatogenesis, in order to elucidate the key stages and cell types that neurl3 plays a role in; (2) Functional analysis of neurl3 in spermatogenesis. RNA interference, gene editing and overexpression technology will be performed to generate gene knockdown, knockout, and overexpression individuals, whose physiological phenotypes will be examined to uncover the role of neurl3 in spermatogenesis; (3) We will employ co-immunoprecipitation and in vitro ubiquitination to screen substrates of Neurl3 protein, and then construct the regulatory network in combination with RNA-seq and co-expression analysis. We can analyze the difference of regulatory mechanism between male and pseudomale spermatogenesis, and thus lay the theoretical basis for the study of sex control in Chinese tongue sole.