Cloning And Molecular Characterization Of Vasa Gene From The Chinese Mitten Crab, Eriocheir Sinensis

The vasa gene encodes an ATP-dependent RNA helicase belonging to the DEAD-box family that is specifically expressed in germline cells in many organisms, first reported as the germ plasm in Drosophila melanogaster. It is widely used as molecular markers of germ cells, mainly for gametogenesis and primordial germ cells (primordial germ cells, PGCs) origin, migration and differentiation, vasa gene plays an important role in animal reproduction and reproductive control. However, the function of DEAD-box proteins in aquaculture species is poorly understood at molecular level.In this study we first cloned Eriocheir sinensis vasa (Es-vasa) full length cDNA sequence by the rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed the cDNA of Es-vasa comprises 2369 bp consists of 48 bp 5'untranslated region (5'UTR),1866 bp open reading frame (ORF) encoding 621 amino acids and 455 bp 3'UTR. The deduced amino acids sequence contains eight conserved motifs belonging to the DEAD-box protein family, GG doublet and Q-motif, zinc-finger motif (Boxed CCHC). The deduced amino acids are encoded DEADc and the HELICc two functional domains. The Es-vasa sequence shows high similarity to VASA homologue of Scylla serrata (77%).Using Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) show that Es-vasa mRNA transcripts were specifically expressed in the germ cells of both male and female adult crabs, not examined in any somatic organs, including the heart, haemocytes, muscle, stomach, intestine, gills, hepatopancreas, thoracic ganglion, cranial ganglion and accessory gonad. Quantitative Real-Time PCR was employed to investigate the tissue distribution of Es-vasa mRNA in ovary and testis of E. sinensis withβ-actin as internal control (P<0.05), showing significant difference in various periods.The Chinese mitten crab (E. sinensis), one of the most commercially important species which is a unique species of crab that is native to China, is a catadromous crustacean with a life-span about two years and have one reproductive season and die shortly after reproduction. Reproduction biology of shrimps and crabs has become one of the major hot spots in crustacean zoology, however, various problems has generally existed in the cultured species populations with the development of intensive culture. Recent years, our laboratory did some works on the molecular biology of reproductive system of E. sinensis, such as cDNA library construction of hepatopancreas and testis, and differentially expressed genes of accessory sex gland by SSH. In the cDNA library of testis, we found a very important gene relevant to regulation of reproduction, the vasa gene regarding the DEAD-box family. In our study, we cloned the full cDNA length of E. sinensis vasa gene (Es-vasa), characterized the mRNA distribution in various tissues and the expression pattern of Es-vasa in different phases of gonadal development using semi-quantitative RT-PCR and quantitative Real-Time PCR analyses. The results show this gene may play a key role and should improve our understanding the molecular mechanism of the regulation of germ-line reproduction and hope to make sure the origin of germ cells determination in E.sinensis in future.