Identification and characterization of Oct4 in Japanese flounder (Paralichthys olivaceus)

• 1、College of Marine Life Science, Ocean University of China

Abstract：In this study, PoOct4 was cloned through homology cloning method and RACE strategy from the blastula-stage embryos. The genomic DNA sequence of Po-Oct4 is 3978 bp long and is consisted of five exons. There exits two transcript isoforms with an identical 5'UTR of 352 bp and different lengths of 3'UTR, which are 874 bp and 1318 bp long, respectively. The deduced Po-Oct4 contained an open reading frame of 1428 bp encoding a protein of 475 residues. The predicted Po-Oct4 possessed a characteristic POU domain that consists of the POU specific domain, the POU homeodomain and a linker region between them. Phylogenetic, gene structure and chromosome synteny analysis provided the evidence that Po-Oct4 is homologous to the mammalian Oct4 gene. We isolated the entire promoter region of Po-Oct4 by genome walking strategy. Sequence analysis with the MatInspector program revealed that there were numerous transcription factor binding sites on the promoter region of Po-Oct4, including Oct 3_4, Nanog, PRDM14, FOXP1-ES, and ONST which may regulate the target gene expression in maintaining pluripotency. Po-Oct4 was observed to be maternally expressed, and the transcripts were present in the unfertilized egg and throughout early development. Po-Oct4 reached its highest expression level at high-blastula stage, decreased but still relatively high until mid-gastrula stage, then greatly diminished at the end of gastrulation stage. From heart-beating stage onward, Po-Oct4 can hardly be detected anymore. Whole mount in situ hybridization (WISH) analyses demonstrated that the transcripts were present in all blastomeres of the early embryo. Po-Oct4 transcripts were exclusively restricted to the gonad tissues among ten examined tissues and with much more abundance in ovary than in testis.

Keywords： Genetics Oct4 Stem cell Japanese flounder