cDNA cloning and expression analysis of a vasa-like gene in Spinibarbus caldwelli

In order to interpret the development mechanism of fish germ cells at a molecular level, a Spinibarbus caldwelli homolog of the zebrafish vasa gene, ScVHG(S. caldwelli vasa homolog gene), was cloned and characterized for use as a molecular marker for germ cells in this species. Analysis of the nucleotide sequence revealed that ScVHG comprises an open reading frame of 1 932 bps encoding 644 amino acids. The deduced amino acid sequence contained nine conserved motifs belonging to the DEAD-box protein family. The S. caldwelli VASA protein sequence showed high similarity to that of zebrafish (77%), Oreochromis niloticus(77%), Oncorhynchus mykiss(79%), Carassius auratus gibelio(90%), Monopterus albus(74%), Carassius auratus(89%) Thunnus orientalis(79%) and Ctenopharyngodon idellus(86%). In adult tissues, the ScVHG transcripts were specifically detected in ovary and testis. In situ hybridization analysis showed that ScVHG messenger RNA (mRNA) was detected in spermatogonia, but not in spermatocytes, spermatoblasts and sperms; During oogenesis, ScVHG mRNA was detected in all time phases from oogonia to oocytes. The earlier phase of oogenesis, the more ScVHG mRNA molecules were detected. The positive signals of ScVHG mRNA gradually weakened in the late phase of oocytes and finally these signals gathered round the cell edge. Consequently, consensus sequences, sequence similarity, and specific localization of ScVHG mRNA in the germ cells all suggest that ScVHG is the S. caldwelli homolog of the zebrafish vasa. Further, ScVHG can be used as a molecular marker for S. caldwelli germ cells.