Dnah3和Tekt1在异源三倍体鲫精子细胞分化中的功能研究

前期研究表明异源三倍体鲫的精子细胞不能形成成熟的正常精子。本项目在获得异源三倍体鲫及其对照鱼的成熟精巢转录本和miRNA数据基础上，将聚焦与精子形成相关的重要基因- Dnah3和Tekt1的结构、表达和功能研究，运用实时荧光定量PCR、免疫组化、CRISPR/Cas9基因编辑技术、精子活力测定、精子细胞分离及孵育等研究手段，系统研究Dnah3和Tekt1基因在异源三倍体鲫精子细胞分化（精子形成）过程中的功能；同时，拟运用双荧光素酶报告基因实验验证异源三倍体鲫表达上调的miR-199-5p与Dnah3和Tekt1基因的靶标关系。该研究不仅有助于阐明Dnah3和Tekt1在鱼类精子形成中的关键作用，还将在分子水平促进揭示异源三倍体鲫的不育机制研究。

Previously research indicated that round spermatids in allotriploid crucian carp did not form mature sperm that possess normal nuclei and integrated tails. In this project, we will pay attention to two key genes related with spermiogenesis, namely, Dnah3 and Tekt1, according to transcripts and miRNAs of mature testes in allotriploid crucian carp and diploid crucian carp. Studies on gene structures, expression and functions of these two genes will be carried out. By using quantitative real-time PCR, immunohistochemical staining, CRISPR/Cas9 knock-out technology, measure of sperm vitality and so on, we are planning to survey the functions of Dnah3 and Tekt1 in spermiogenesis of allotriploid crucian carp. Meanwhile, relationship between miR-199-5p and Dnah3 or Tekt1 will be tested by using the dual luciferase reporter assay system. This study will not only elucidate the key roles of the Dnah3 and Tekt1 genes during spermiogenesis in fish, but also promote studies on mechanism on sterility in allotriploid crucian carp at molecular level.