Molecular Cloning And Expression Analysis Of Glutathione S-transferase In The Freshwater Bivalve Cristaria Plicata

The freshwater bivalve Cristaria plicata(Unionidae),which known as “pearl bivalves” of China.It is easy to be infected to cause diseases and increase the risk in the breeding industry.Therefore,it is of great significance to study the molecular immunity of shellfish.In this paper,the cDNA sequences of Mu-class and Theta-class glutathione S-transferase(GST)genes were cloned by nested PCR and RACE PCR.The full-length cDNA of CpGST3 is 1026 bp.It contains an open reading frame of 558 bp and encodes 185 amino acids,with a 5 bp non-coding region of 71 bp and a3’ non-coding region of 397 bp.The molecular weight of the protein is 21.7 kDa.The isoelectric point of the protein is 5.38.The full-length cDNA of CpGST4 is 1301 bp,which contains an open reading frame of 759 bp,which can encode 252 amino acids,185 bp in the 5’ non-coding region and 357 bp in the 3’ non-coding region.The protein molecular weight is 29.30 kDa,the isoelectric point is 6.17.Through the analysis of the expression patterns of CpGST3 and CpGST4 genes in five tissues of pupa,we found that CpGST3 and CpGST4 genes were expressed in iliac crest tissue,hepatopancreas,adductor muscle,mantle,and hemolymph,with the highest expression of CpGST3 gene.The hepatopancreas was the lowest in the sputum,and the highest expression of CpGST4 was also in the hepatopancreas,but the mantle was the lowest.The expression of CpGST3 and CpGST4 in the haemolymph and hepatopancreas of Cristaria plicata after stimulation with microcystin was detected.The results showed that CpGST3 in sputum blood cells and hepatopancreas were stimulated by microcystin to stimulate sputum.The amount of gene expression began to rise,reaching the highest at 24 h and 12 h,and then gradually decreased.The expression level of CpGST4 gene also gradually increased.The expression level reached the highest at 12 h and then gradually decreased.The open reading frame of CpGST3 and CpGST4 genes and the PET-32 a plasmid were also digested with HindIII and Bam H1,and the PET-32a-CpGST3 and PET-32a-CpGST4 prokaryotic expression plasmids were successfully ligated and constructed.The recombinant plasmid was transferred into BL2 and induced by IPTG to obtain fusion protein.The results showed that the recombinant protein existed inthe form of inclusion bodies and purified with a purification column to obtain a concentration of 0.4676 mg/mL.CpGST3 fusion protein and 0.312 mg/m L of CpGST4 fusion protein.