Melecular Cloning And Expression Analysis Of GST From Cristaria Plicata

The freshwater mussel(Cristaria plicata), which is of great economical importance and known as “pearl bivalves” in the aquaculture industry of China, has been suffering serious problemsdue to the outbreak of diseases. Thus, understanding of the immunity of reshwater mussel is crucial fordiseases management and development of sustainable musselculture and pearl production.In this study, sigma-class glutathione S-transferase(CpGST1) and Sigma-class glutathione S-transferase(CpGST2) cDNA were cloned from C. Plicata using rapid amplification of c DNA ends and nested PCR. The full-length cDNA sequence of CpGST1 was 1610 bp, the 5’untranslated region(UTR) and 3’UTR was 320 bp and 600 bp respectively, and contained a 690 bp open reading frame(ORF) coded for a 230-amina acids. The calculated molecular mass and isoelectric point(PI) of deduced protein were 25.8 KDa and 8.87, respectively. SignalP program analysis showed that it had no a signal peptide. The length cDNA sequence of CpGST2 was 862 bp, the 5’untranslated region(UTR) and 3’ UTR was 151 bp and 33 bp respectively, and contained a 642 bp open reading frame(ORF) coded for a 214-amina acids. The calculated molecular mass and isoelectric point(PI) of deduced protein were 23.9KDa and 6.67, respectively.SignalP program analysis showed that it is not a signal peptide.The expression of CpGST1 and CpGST2 mRNA in C. plicata were measured. The results showed that both of mRNA of CpGST1 and CpGST2 were express in tissues, but had significant differences in hemocytes, hepatopancreas, muscle, mantle, gills. The expression level of CpGST1 and CpGST2 in hemocytes was the lowest, followed by the muscle and mantle, the expression level of CpGST1 and CpGST2 in hepatopancreas was the highest. After A. hydrophila stimulation, the expression of these two genes had significant in tissues of hepatopancreas and hemocytes.The Product of gene amplification was transferred to the expression vector of PET-32 a after HindIIIand XhoI double enzyme, and the prokaryotic expression plasmid of PET-32a-GST2 was successfully builded. The plasmid was transferred into Rosetta-gami(DE3) of E. coli, The concentration was 1mmol/L with added IPTG at, 37 ℃ 8 h. The fusion protein was induced by Ni2+ affinity chromatography column purification, and the activity of the purified protein was 46.965±0.082 μmoL/min /mg。...