半滑舌鳎tesk1和neurl3基因在精子发生过程中的功能研究

在半滑舌鳎养殖中，伪雄鱼W精子缺失导致无法产生超雌鱼后代，因此对精子发生过程中相关功能基因的研究是利用伪雄鱼育种提高雌鱼比例的关键。前期工作筛选到的两条Z染色体特异基因，tesk1和neurl3，为研究W染色体缺失基因与W精子缺失之间的关系提供了理想模型。本课题将从以下四个方面进行研究：（i）以不同发育阶段的性腺为材料，通过RT-PCR和Western blot研究其时间表达模式；（ii）对高表达时期的性腺进行石蜡切片，通过原位杂交和免疫组化研究其在不同生殖细胞中的分布；（iii）注射siRNA和重组蛋白对基因进行抑制或模拟过量表达，探索基因剂量与精子发生过程的关系；（iv）免疫共沉淀寻找互作蛋白，初步建立精子发生过程中的蛋白互作网络。本研究将详细探索半滑舌鳎精子发生相关基因的功能，为阐明伪雄鱼中W精子缺失机制，提高精子质量提供理论基础。

In Cynoglossus semilaevis culture, the W-sperm absence of pseudomale results in the nonexistence of super-female offspring. Therefore, it is necessary to investigate the functional genes involved in the spermatogenesis for application of pseudomale breeding, in order to improve the female proportion. Based on our previous work, two Z chromosome linked genes, tesk1 and neurl3, were identified as ideal models to reveal the relationship between W-chromosome-lacking genes and W-sperm absence. This study will focus on the following four aspects: (i) Temporal expression patterns of tesk1 and neurl3 using RT-PCR and Western blot in gonads from different developmental stages. (ii) Spatial expression patterns in specific germ cells using in situ hybridization and immunohistochemistry in paraffin sections of gonads at high-expression stages. (iii) Dissecting the relationship between gene-dosage and spermatogenesis after injection of siRNA and recombinant proteins to inhibit gene expression or mimic overexpression. (iv) Search for interacting partners via co-immunoprecipitation and establish preliminary interaction networks during spermatogenesis. In this study, the functionality of spermatogenesis-related genes will be elucidated in detail, thereby providing a fundamental basis to cope with the problem of W-sperm absence in pseudomale and further improve the sperm quality.