Identification And Expression Of Glutathione Peroxidase 1 And Glutathione Peroxidase 4 From Larimichthys Crocea

In this paper,based on the whole genome database of Larimichthys crocea,we cloned the full-length cDNA of LcGPxla,LcGPxlb,LcGPx4a and LcGPx4b and analyzed their sequence structures and transcript abundance in different tissues.Additionally,by means of peritoneal infection of pathogenic Vibrio parahemolyticus,the real-time quantitative PCR technology are used to analyze the relative expression levels of LcGPx1a,LcGPx1b,LcGPx4a and LcGPx4b mRNA in four major immune-related tissues,including liver,spleen,kidney and head kidney.Meantime,we chosen LcGPx1a and LcGPxlb genes to construct their prokaryotic expression system and measure their enzymatic activity.Results were shown as follows.The partial sequences of LcGPxla,LcGPxlb,LcGPx4a and LcGPx4b were obtained from the whole genome database of L.crocea.By virtue of method of 3’,5’Rapid Amplification of cDNAEnds(RACE),we got their full-length cDNA sequences,respectively,and made analyses for these sequence structures.Sequence analysis revealed that the full-length cDNA sequences of LcGPxla,LcGPxlb,LcGPx4a and LcGPx4b were 917 bp,884 bp,1149 bp,and 1161 bp,respectively.Selenocysteine insertion elements(SECIS)identified in both their 3’-untranslated region sequences on the SECISearech website,the result indicated that SECIS elements of LcGPxlb were type II and the others belonging to type I SECIS elements.According to the analysis of their amino acids sequences on the InterPro Scan 5 and SingalP websites,we found that both of themhad the specific codon UGA encoding selenocysteine(Sec)and the catalytic tetrad(Sec,Gln,Trp,and Asn)which was the unique structure of GPx family.Moreover,the amino acid sequence of LcGPx4b contained an N-terminal signal peptide sequence consisting of 15 amino acids but other members didn’t have any signal peptide sequences.At last,the GPx phylogenetic tree was constructed by Mrbayes v3.2.6 software.It revealed that the fish GPxl and GPx4 branches could be divided into the fish GPxla,GPx lb,GPx4a and GPx4b branches,which indicated that these two GPx members might be have multiple isoforms in fish.Such results demonstrated that four members’ sequence characteristics and amino acid structure composition,which laid a molecular theoretical basis for subsequent functional studies.The expression patterns of LcGPxla,LcGPxlb,LcGPx4a and LcGPx4b in L.crocea were studied by real-time quantitative PCR.The results shown that both members were widely expressed in different tissues.To be specific,transcript level of LcGPxla was not significantly different among tissues,whereas that of LcGPx1b was higher in the kidney and head kidney than in other tissues,while that of LcGPx4a and LcGPx4b was higher in the liver than in other tissues.After Vibrio parahemolyticus invasion,the temporal expression profiles of LcGPx1a,LcGPx1b,LcGPx4a and LcGPx4b were investigated by real time PCR technology in four major immune-related tissues(liver,spleen,kidney,and head kidney).The results illustrated that four members had different expression patterns,respectively,during the Oh-96h period.The most remarkable change of LcGPx1a and LcGPx1b genes occurred in the liver.The relative expression level of LcGPx1a was downregulated but that of LcGPxlb was upregulated during the experiment period.Their relative expression levels reached the lowest and the highest value at 24h post-injection,respectively,compared with that in the control group.And that of LcGPx4a and LcGPx4b were both up-regulated in spleen during the experiment period.Results above mentioned revealed that the relative expression levels of LcGPx1a and LcGPx1b had significantly changed against V.parahemolyticus,so these two members were chosen to construct their prokaryotic expression system and verify the antioxidant capacity.Firstly,in LcGPx1a and LcGPx1b sequences,the single-base mutation method was used for mutating the UGA codon encoding of the Sec into UGC codon encoding of the cysteine(Cys).Then the Cys mutant pET-tla and pET-tlb plasmids were constructed and expressed in E.coli BL21(DE3).After purified by Ni-NTA column,dialyzed and concentrated method,the recombinant proteins tla and tlb were obtained,respectively.And their enzymatic activity was measured by using the total glutathione peroxidase detection kit.The enzyme activity of tla was 414.09 ±42.35 mU/mg and that of tlb was 71.43 ± 8.25 mU/mg.Therefore,tla maybe have stronger antioxidant capacity than tlb.In conclusion,all above results in this paper indicated that the sequence structures of LcGPx1a,LcGPx1b,LcGPx4a and LcGPx4b and their temporal expression profiles during the anti-bacterial immune period.And these results would provide several basis foundations for the further study of the functional differentiation of GPx in L.crocea.