Cloning And Prokaryotic Expression Of Vasa Gene From The Freshwater Crab Sinopotamon Henanense

The vasa gene encodes an ATP-dependent RNA helicase,which belongs to the DEAD-box family,and was first reported in Drosophila.It is specifically expressed in germline cells,therefore,it is used as a molecular marker of germ cells,mainly for gametogenesis and primordial germ cells specification and migration.Thus,we can consider that vasa plays an important role in animal germ cells development and reproductive regulation.Nucleotide sequence analysis revealed the cDNA of Sh-vasa comprises 2896bp,1329bp translated region,encoding 442 animo acids.Blast results showed that the sequence we obtained have high similarity in amino acid sequences and protein sequences of cDNA with fishes,birds and mammals and some invertebrates.Motif analysis showed that the deduced amino acid sequence contains four conserved motifs(motif??motif la? motif?b?motif?)belonging to the DEAD-box protein family,GG doublet,Q-motif,zinc-finger motif,RGG repeat,and glycine-rich regions.However,DEAD-box family protein has eight conserved motifs,motif??motif la?motif?b?motif??motif??motif??motif??motif?,and two zinc-finger motifs,Q-motif,GG doublet,At that,the animo acids sequence we obtained is correct in front of motif?(including motif?).Behind that,protein translation terminates early.Therefore,the 3' end of cDNA of Sh-vasa that we cloned needs further validation.Multiple sequence alignment and phylogenetic analysis showed the Sh-vasa have high similarity to VASA homologue of shrimps and crabs.Above all indicated that the cDNA sequence we obtained in this study,is the 5' end of vasa cDNA sequence of the freshwater crab Sinopotamon henanense.Semi-quantitative PCR(Sq-PCR)analysis confirmed the distribution situation of Sh-vasa in different kinds of organizations of S.henanense,the results showed that vasa gene of S.henanense also specifically expressed in germline cells.Based on the cDNA sequence of Sh-vasa,we designed two pairs of primers,used PCR amplification,added EcoR I and Xho I restriction sites to clone vasa gene at both ends.The vasa gene was cloned into pET-28a,pET-32a by recombinant DNA technology.We constructed four expression vectors,Vexp-28a?Vexp-32a?Ve-28a and Ve-32a successfully and induced with IPTG for prokaryotic expression.After purification,we analysed the proteins by SDS-PAGE and Western blot,the results showed that the protein is not VASA fusion protein of S.henanense,we need to improve the experimental schemes,then do experiments again,in order to obtain the VASA fusion protein of S.henanense.