猪BAG6可变剪接体鉴定及其在精子发生中的功能研究

选择性剪接在动物精子发生等过程中起重要调控作用。申请人前期通过对性成熟前后大白猪睾丸组织RNA-seq，分析其选择性剪接事件，发现BCL2相关抗凋亡基因6（BAG6）出现外显子跳跃而导致蛋白截短，且该剪接体在性成熟前猪睾丸组织中显著高表达。此外BAG6敲除会出现减数分裂期的小鼠精子细胞凋亡，导致雄性完全不育。然而猪BAG6可变剪接体及其在精子发生过程中的功能和相关机制尚不清楚。有鉴于此，本项目首先鉴定猪BAG6可变剪接体，检测其时空表达谱；然后在猪睾丸ST细胞中，通过抑制表达、超表达等研究猪BAG6可变剪接体功能；再综合利用双荧光报告系统、定点突变、免疫共沉淀、酵母双杂交等，鉴定猪BAG6剪接体的顺反式调控因子、互作蛋白及结合位点；最后通过睾丸组织条件性敲除小鼠模型，从活体水平较系统地研究BAG6可变剪接体参与精子发生的机制和调控网络。本项目研究为猪精子发生的遗传调控提供理论基础和新见解。

Alternative splicing plays an important role in the process of animal spermatogenesis. In our previous study, RNA-seq of the sexually mature and immature testes from Large White pigs have been done. The alternative splicing event analysis of RNA-seq data showed that BAG6 emerged exon skipping, leading to protein truncation. Exon-skipped BAG6 transcript was up-regulated in immature testes. Furthermore, inactivation of BAG6 in mice resulted in widespread apoptosis of meiotic male germ cells and complete male infertility. However, it still remains unknown on the characteristics, functions and the relative genetic mechanism of porcine BAG6 splice variants in spermatogenesis. In this proposal, we will identify porcine BAG6 splice variants and detect their spatiotemporal expression profiles, and study the effect of porcine BAG6 splice variants on spermatogenesis through suppressed-expression and over-expression in porcine ST cell, then identify cis-trans regulatory factors and interacting proteins of porcine BAG6 splice variants by dual luciferase reporter system, site-directed mutagenesis, co-immunoprecipitation, and yeast two-hybrid system, and finally investigate the effect of porcine BAG6 splice variants on spermatogenesis in vivo via testicular -specific BAG6 splice variants knockout mice model. We expect to systematically investigate the essential role of porcine BAG6 splice variants in spermatogenesis and to establish the related genetic network in vitro and in vivo. This proposal will provide a basic foundation and a new insight into the genetic regulation of spermatogenesis in the boar.