A Preliminary Function Analysis Of Dead End Gene From Olive Flounder (Paralichthys Olivaceus)

During the life cycle of fish, the primordial germ cells(primordial germ cells,PGCs) not only able to reproduce through generating sperm and egg cells, but also plays an important role in the sex differentiation process. Therefore, research on fish primordial germ cell is important for fish resource protection, reproduction and sex regulation. The olive flounder(Paralichthys olivaceus) is one of the important marine economic fish, research on the PGCs developmental regulation mechanisms will provide new ideas for the resources protection, genetic breeding and regulation of sex differentiation. It is a prerequisite to get PGC development related gene and study its function for the application of PGC in flounder. In this study, flounder PGC marker gene dead ene(dnd) was cloned, and its expression and function was analysis too.We cloned flounder PGC related gene dnd and found there was five dnd isoforms:dnda, dndb, dndc, dndd, dnde. The code sequecne of Dnda contained the complete Dnd conserved domains: one NR(N-terminal Region), one RRM(RNA recognition motif) and four CR(C-terminal Region) domains, while the other four missing one or more domain.Sequence alignment and phylogenetic analysis showed that flounder Dnd grouped particularly with turbot Dnd in a branch.The expression pattern of dnd during flounder embryonic development of gonadal development and differentiation was analysis through RT-PCR. The results showed that flounder different isoforms of dnd show different expression pattern,some dnd isofroms was continuously expressed and the expression of some dnd isofroms decreased during embryonic development. During gonad development and differentiation process, the expression of dndb was lower than that of other isoforms and dndb wasn’t expressed in the I phase and IV phase of ovaries development and I phase of testis development. The expression of dndc was not expressed in the I phase of testis. And the results of in situ hybridization indicated that all dnd was expressed specifically in the PGCs during embryonic development.Dnd-GFP, recombinant expression vector, was constructed and expressed in HeLa cell to test the subcellular localization of different dnd isoforms. Dnda, Dndcand Dndd located in the nucleus and cytoplasms, whereas Dndb and Dnde were only expressed in the nucleus. Thus, RRM, and NR and CR1 domain of Dnd might be nuclear localization and cytoplasmic localization signal peptide, respectively.Eukaryotic recombinant protein expression vectors were constructed and used to express Dnd different isoforms in HEK 293 T cells. EMSA was used to detect the interaction between Dnd and nanos3 3’UTR.The results showed that Dnda, Dndc,Dnde could bind nanos3 3’UTR, while Dndb andDndd could not.dndc-dsRNAi was synthesized and injected in juvenile flounder(about 2.0cm) to explore the function of dndc. Results showed knockdown dndc could promote the growth of juvenile fish. It might be that knockdown dndc inhibited PGC development,and the material and energy for PGC were used for growth.