Cloning And Expression Of Vasa And β-actin Gene From Urechis Unicinctus

VASA protein is a member of the DEAD(Asp-Glu-Alu-Asp) box family of ATP-dependent RNA helicases and initially found as a component of germ plasm in Drosophila. vasa gene is specifically expressed in germ-line cells of many metazoans. At present, vasa gene has been extensively used as a specific molecular marker of germ-line cells and played an important role in the research of gametogenesis and reproductive regulation. Urechis unicinctus, classified to Echiuroidea, is a familiar species of echiuran along the coast of Yellow and Bohai in China. In the present study, homolog cloning strategy and SMART RACE technique were used to isolate and clone the vasa gene of U. unicinctus, and semi-quantitative RT-PCR was conducted to analyze the spatial and temporal expression of vasa mRNA. The results are expected to provide some basic knowledge for further investigation of ontogenesis, differentiation of germ cells and reproduction regulation in U. unicinctus.The full length vasa cDNA sequence of 4,080 bp comprised an open reading frame (ORF) of 2,322 bp encoding a polypeptide of 773 amino acids, a 5'untranslated region (UTR) of 322 bp and a 3'UTR of 1,436 bp. The deduced amino-acid sequence showed a great similarity to VASA homologues in mammal, fish, amphibian, insect and other invertebrate. Besides eight conserved motifs of all DEAD-box family proteins were observed, the U. unicinctus VASA protein shared multiple arginine-glycine-arginine (RGG) repeats and a glycine(G)-rich in the NH2-terminal portion with other VASA-related proteins. Furthermore, phylogenetic analysis showed the deduced amino acids sequence was classified to VASA-related proteins, and was designated as UuVASA. The spatial expression analysis of vasa mRNA showed a high level of transcripts in germ cell both in females and males,...