Cloning Of Vasa Gene In Japanese Flounder (Paralichthys Olivaceus) And Construction Of GFP Reporter Vectors Driven By Vasa Promoter

Primordial germ cells (PGCs) are precursors of germ cells that migrate from the siteof origin to the gonadal primordia and eventually give rise to sperm and egg in sexuallyreproducing organisms. Vasa gene provides the most reliable molecular marker forstudies on the germ cell development and reproductive regulation. Japanese flounder(Paralichthys olivaceus) is one of the most important marine economic fish in the coastof East Asia. It is significant to isolate its PGCs for culture, cryopreservation andtransplantation of germline stem cells. In this study, the full-length of the vasa gene wascloned from Japanese flounder. Then the expression file in different tissues and earlydevelopmental stages was demonstrated. After cloning of promoter region of Po-vasa,the GFP reporter vector (pvasa-EGFP) driven by Po-vasa promoter was constructed totrace and isolate PGCs in the future.The full-length Po-vasa cDNA was2461bp, composed of175bp of5UTR,175bpof ORF, and1941bp of3UTR, and it could encode646amino acid residues. Theputative amino acid sequence shared all the nine conserved motifs charaeteristic andRG-or RGG-repeats in N-terminal of the DEAD-box family. In the11.4kb genomicsequence,23exons were identified besides3333bp5and1183bp3flanking regions.The start codon of Po-vasa was in exonII. The5UTRof Po-vasa was encoded by exonI and a small part of exon II. Twelve small exons (include the remaining part of exon II)encoded the RG-and RGG-rich N-terminal region, while the other ten larger exonsconsisted the rest of the sequences that include the nine consensus motifs of theDEAD-box family.Quantitative real-time PCR analysis showed that vasa gene expression was restrictedto the testis and ovary in adults, with a higher level in the ovary. The Po-vasa mRNAwas found at all early embryo development stages including unfertilized eggs,indicating the maternal deposit of vasa. The expression level of vasa remainedrelatively high after fertilization, with a slight increase and decrease before gastrula. During gastrula, its expression descended steeply to a low level, which maintaineduntil1day after hatching, the last stage studied.During the analysis of the PoVasa transcripts, a total of160clones were sequencedfrom male and female tissues, respectively, from which ten different5extremities wereidentified. Thus, ten different isoforms of vasa mRNA, derived from the5alternativesplicing of the same gene, were identified and characterized. Isoform A containing allthe23exons was the first determined and the predominant one among all isoforms. Inisoform B and C, exon II containing the initiator codon ATG was deleted and thustranslation could not be started until exon VIII where an alternative ATG codon could befound. Most of these isoforms contained the eight consensus motifs of the DEAD-boxfamily without changing the reading frame except isoform J that showed frame shift dueto a deletion of8nucleotides. The predominant proportion of transcripts was isoform A.The diversity of N-terminal region indicated high variability in vasa mRNA processingin Japanese flounder. Relative quantity of isoforms B~J showed differences in gonads.E and F both had a higher level in ovary than in testis. Besides, these isoforms did notshare a same expression pattern. These results indicated possibility of differentfunctions of these divers isoforms.Through cis-acting analysis by a series of bioinformatics software, a characterizeTATA box was found at31bp upstream from the transcription start site (TSS).Putative binding sites of several transcription factors, including GATA-1、C/EBP、AP-1、SP-1、Oct-1、MZF1、c-Myc、SRY、Sox-5、AML-1a、HNF-3B, were foundin5flanking region. Several sequences binding sites of glucocorticoid receptor (GR)were also identified in the promoter of Po-vasa. These TF may have the function ofregulating vasa expression.EGFP gene sequence and5and3regulatory regions of Po-vasa gene were allinserted in a vector. Thus a reporter vector pvasa-EGFP was constructed and laid thegroundwork for identifying fish primordial germ cells (PGCs) and investigating germcell biology.