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

piwi gene is a regulatory reproductive gene and belongs to the PIWI subfamily of Argonaute family.It was first identified in Drosophila melanogaster which played an important role in the self-renewal of germ stem cells.piwi gene is specifically expressed in germ cells.It functions with piRNA form PIWI-piRNA pathway to maintain stability of germ Line system and ensure normal gametogenesis reproduction.In this study,based on the transcriptome sequencing and partial sequences of piwi in previous,the main results are as follows:(1)we cloned the full-length cDNA sequence of piwi gene from Sinopotamon henanense(Shpiwi).Shpiwi included 2757 bp ORF,36 bp 5'UTR and 1127 bp 3'UTR,and encoded 918 amino acids.The molecular weight of protein is103.804 KDa.(2)Blast P analysis showed that the protein sequence have high homology with the amino acid sequences from Eriocheir sinensis,Penaeus monodon and Portunus trituberculatus.Protein domain analysis showed that Sh PIWI existed RG/ RA motifs catalyzed by PRMT5 in the Nterminal domain and four concerved domins : N-terminal domain ? PAZ domain?MID domain and PIWI domain.(3)Furthermore,to study the function of the PIWI protein,the prokaryotic expression of Sh PIWI protein in S.henanense was performed.After the expression sequence of Shpiwi was cloned into expression vector,and the prokaryotic expression vector was successfully constructed.By optimizing the induced expression conditions and SDS-PAGE detection,and the results showed that the best induction condition is 1 m M IPTG at 24 ? for 12 h.it was confirmed that we obtained large number of the PIWI fusion protein in the form of inclusion body.These results provide a basis on further study the molecular mechanism of reproductive toxicity in crustacean.