Characteristic And Functional Analysis Of Prdm14 And Prdm1 Genes In Japanese Flounder (Paralichthys Olivaceus)

The Japanese flounder (Paralichthys olivaceus) is an important farmed marine fish in our country. Researchers had been trying for year to acquire good varieties, however how to protect the genetic resources of the selected varieties was a problem that needed to be solved immediately. With the development of stem cell research, fish embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and primordial germ cells (PGCs) drive people's attention. Prdml4 played significant role in specification of PGCs, which could be used to protect genetic resources. PGC could be reprogrammed into embryonic germ cells (EGCs) by culture. Since prdm14 played significant roles in maintain stem cell property, otherwise prdm14 and prdml are key transcriptional regulators for PGC specification. A deep research in Japanese flounder prdml4 and prdml is critical for protect genetic resource, so as to laying a foundation for the further investigation.The genomic DNA sequence of Po-prdml4 is 5881 bp long and is consisted of nine exons.The cDNA of Po-prdm14 with 214 bp 5'UTR,576 bp 3'UTR and 1833 bp of the entire open reading frame. The predicted amino acid sequence of Po-prdml4 is 610 residues long and contain a PR domain and six zinc fingers. Peptide sequence alignment, gene structure analysis, and phylogenetic analysis revealed that Po-PRDM14 was homologous to mammalian PRDMM. We isolated the entire promoter region of Po-prdm14, sequence analysis prognosed that multiple transcription factors (TFs)bind to the 5'regulatory region of Po-Prdm14, these factors related to the function which reported by mammalian prdm14 before. These functions are PGC specification, maintain stem cell property and so on. Results of real-time quantitative polymerase-chain-reaction amplification and in situ hybridization in embryos demonstrated thatPo-prdm14 was highly expressed between the morula and late gastrula stages, with its expression peaking in the early gastrula stage. Relatively low expression of Po-prdml4 was observed in the other developmental stages. In situ hybridization of gonadal tissues revealed that the transcripts were located in the nucleus of the oocytes in the ovaries but only in the spermatogonia and not the spermatocytes in the testes.The Po-prdml is consisted of seven exons.The cDNA of Po-prdml with 128 bp 5'UTR,1746 bp 3'UTR and 2532 bp of the entire open reading frame. The predicted amino acid sequence of Po-prdml is 843 residues long and contain a PR domain and five zinc fingers. Peptide sequence alignment, gene structure analysis, and phylogenetic analysis revealed that Po-PRDM1 was homologous to mammalian PRDM1. We isolated the promoter region of Po-prdm1, sequence analysis prognosed that multiple TFs bind to the 5'regulatory region of Po-Prdml, these factors related to immunologic function, sexual development and so on. Results of real-time quantitative polymerase-chain-reaction amplification and in situ hybridization in embryos demonstrated that Po-prdml was observed to be maternally expressed, and the transcripts were present in the unfertilized and throughout early development. Po-prdm1 reached its highest expression level at mid-gastrula stage. Po-prdml transcripts were ubiquitously expressed in tissues of adults. In situ hybridization of gonadal tissues revealed that the transcripts were located in the nucleus and cytoplasm of the oocytes in the ovaries but only in the spermatogonia and not the spermatocytes in the testes.