TSSK激酶介导的磷酸化修饰在精子发生学中的生物学功能及作用机制

睾丸组织中精子发生发育过程是一个高度复杂有序而又保守的细胞分裂与分化的过程，大量蛋白质翻译后修饰（PTMs）如乙酰化、泛素化等在精子发生学中起着重要的作用。而磷酸化修饰在精子发育中是否起关键作用尚不知晓。项目前期在果蝇中鉴定到一个在睾丸组织中大量表达的激酶（dTSSK），该基因的敲除导致雄性不育，初步推测该激酶诱导的磷酸化过程可能参于精子发生发育过程。项目拟结合CRISPR基因打靶与phiC31重组技术，构建dTSSK敲除突变体和一系列功能补偿突变体，系统的研究dTSSK的亚细胞定位，鉴定该激酶缺失后精子发育的缺陷表型，系统的探索dTSSK在精子发生学中的生物学功能，并通过磷酸化蛋白质组质谱技术鉴定dTSSK修饰的所有磷酸化底物。通过一系列体外体内实验和遗传功能验证最终阐明dTSSK介导的磷酸化修饰在精子发生学中的分子调控网络。

Spermatogenesis in testis is a highly complex, ordered and conserved process of cell division and differentiation. A large number of protein post-translational modifications (PTMs), such as acetylation and ubiquitination, play an important role in spermatogenesis. However, whether phosphorylation plays a key role in sperm development is unknown. In the early stage of the project, a kinase (dTSSK), which was exclusively expressed in testis, was identified, whose knockout mutation caused male infertility. Thus, it is speculated that dTSSK induced phosphorylation may be involved in Drosophila spermatogenesis and play a key role in sperm development. The project intends to combine CRISPR gene targeting and phiC31 recombination technology to construct dTSSK knockout mutants and a series of functionally compensatory mutants, systematically study the subcellular localization of dTSSK, identify the defective phenotype of sperm development after dTSSK deletion, systematically explore the biological functions of dTSSK in spermatogenesis, and identify potential phosphorylation substrates of dTSSK through phosphor-proteomic mass spectrometry. Taken together, through a series of in vitro and in vivo experiments and genetic function verification, the molecular regulatory network of dTSSK-mediated phosphorylation in spermatogenesis will be finally elucidated.