Cloning and Expression Profiling of the Gene vasa during First Annual Gonadal Development of Cobia (Rachycentron canadum)

The vasa gene is essential for germ cell development and gametogenesis both in vertebrates and in invertebrates. In the present study, vasa (Rcvasa) cDNA was cloned from cobia (Rachycentron canadum) using the RACE amplification method. We found that the full-length cDNA sequence of Rcvasa comprises 2571 bp, containing a 5 '-UTR of 145 bp, a 3 '-UTR of 341 bp, and an open reading frame (ORF) of 2085 bp, encoding a protein of 694 aa. The deduced amino acid sequence contains 8 conserved motifs of the DEAD-box protein family, 7 RGG repeats, and 10 RG repeats in the N-terminal region. Comparisons of the deduced amino acid sequence with those of other teleosts revealed the highest percentage identity (86.0%) with Seriola quinqueradiata. By using semiquantitative RT-PCR, Rcvasa appeared to be specifically expressed in the testis and ovary, among 13 tissues analyzed. In addition, annual changes in Rcvasa expression levels were examined in the gonads by quantitative real-time PCR (qRT-PCR). The expression of Rcvasa in the testis first increased significantly at 120 dph (stage II-III), then stabilized as the testis developed from 185 dph (stage III) to 360 dph (stage V). During the development of the ovary (stage I to II), the expression of Rcvasa first increased and reached the highest level at 210 dph (stage II), then decreased. Furthermore, the results of chromogenic in situ hybridization (CISH) revealed that Rcvasa mRNA was mainly expressed in germ cells and barely detected in somatic cells. In the testis, Rcvasa mRNA signal was concentrated in the periphery of spermatogonia, primary spermatocytes, and secondary spermatocytes and was significantly weaker in spermatids and spermatozoa. In the ovary, Rcvasa mRNA signal was uniformly distributed in the perinuclear cytoplasm and was intense in early primary oocytes (stage I and II). These findings could provide a reference for understanding the regulatory mechanisms of vasa expression during the development of germ cells in cobia.