The Study Of Vasa Alleles Mutants And Vasa Interacting Proteins And RNAs In Zebrafish

Germ cells(eggs or sperm)are the carriers of intergenerational heritage information.Eggs and sperm combine to form fertilized eggs and then develop into individuals,and sexually mature individuals produce germ cells again to complete the reproductive cycle.The germline of animals originates from the specialized primordial germ cells(PGCs)in the early embryonic stage.PGCs migrate to the gonads and finally differentiate into sperm or eggs.Zebrafish PGCs contain germplasm,which contains specific RNAs and protein.Maternal factor Vasa is one of the components of the germplasm,and plays a vital role in the reproductive development such as the specialization,proliferation and maintenance of germ cells.Although there is a certain understanding of Vasa,the proteins and RNAs that bind to it still need to be studied in depth.This thesis mainly studies the interacting protein and RNAs of zebrafish Vasa.First,we used CRISPR/Cas9 technology to construct a vasa alleles mutant with a 713 bp fragment(including the start codon)deletion,which provides conditions for in-depth study of Vasa.At the same time,a mouse Vasa antiserum was prepared by expressing the C-terminal peptide of Vasa in vitro as an antigen.The results show that the prepared antiserum can immunoprecipitated endogenous Vasa protein.Using this antiserum for immunoprecipitation and RNA binding protein immunoprecipitation experiments,we found that in the Vasa protein complex of zebrafish stage I oocytes and testes,there are multiple proteins such as Tdrd1,Tdrd6,Tdrd7,Myh9,Myh10;and in the Vasa protein-RNA complex of stage I oocytes,there are a variety of reproductive development-related RNAs such as nanos3,buc,dazl,dnd,pactin,piwi,brul.And it was confirmed in vitro that Vasa can bind to nanos3 3'UTR and vasa 3'UTR.This result suggests that Vasa may play a role in the germ cells development of zebrafish by binding to these proteins and RNAs.Immunofluorescence experiment confirmed that Vasa in zebrafish gonads is mainly expressed in the cytoplasm of stage I oocytes and spermatocytes.In short,these results provide new clues and conditions for further research on the function of Vasa in the reproductive development of zebrafish.