CDNA Cloning And Expression Patterns Of 2-Cys Prxs In Eriocheir Sinensis

The Chinese mitten crab,Eriocheir sinensis,is one of the most important economical aquatic species in China.In recent years,due to the rapid development of inordinate culture,the deterioration of the environment and the challenge by various pathogens,the survival of E.sinensis has been challenged which caused huge economic loss in commercial crab culture.It is of great significance to understand the innate immune response of E.sinensis and explore the diseases prevention and control system.Peroxiredoxins(Prxs)are large families of antioxidant proteins ubiquitously found from prokaryotes to eukaryotes which can maintain the homeostasis between ROS and antioxidants.In this study,the typical and atypical 2-Cys Prx of E.sinensis were cloned and characterized.Better understanding of the innate immune defense system of E.sinensis will be beneficial to the development of health management and disease control in crab culture.In this study,based on the transcriptome data in our laboratory,four isoforms of Prxs genes were cloned from hepatopancreas of E.sinensis by using RACE methods,named EsPrx,EsPrx3,EsPrx4,and EsPrx5,respectively.Bioinformatics analysis was used to analyze the molecule characteristics.Quantitative real-time PCR was used to analyze the tissues distribution and the temporal expression of these genes after Aeromonas hydrophila challenge and the activity changes of Prxs was performed by enzyme-linked immunosorbent assay(ELISA).Meanwhile,four recombinant plasmids were obtained and expressed in E.coli BL21(DE3).The purified recombinant proteins of EsPrx,EsPrx3,EsPrx4 and EsPrx5 were used to detecte the antioxidant properties against H2O2.The results of bioinformatics analysis reveal that the full-length cDNA sequence of EsPrx was 1663 bp,containing an open reading frame(ORF)of 597 bp,encoding 198 amino acids.The predicted molecular weight(MW)was 22.05 kDa and the theoretical isoelectric point(PI)was 5.67;the full-length cDNA sequence of EsPrx3 was 2516 bp,containing an ORF of 681 bp,encoding 226 amino acids.The predicted MW was 24.81 kDa and the theoretical PI was 6.09;the full-length cDNA sequence of EsPrx4 was 1444 bp,containing an ORF of 738 bp,encoding 245 amino acids.The predicted MW was 27.31 kDa and the theoretical PI was 5.55;the full-length cDNA sequence of EsPrx5 was 1252 bp,containing an ORF of 552 bp,encoding 183 amino acids.The predicted MW was 19.67 kDa and the theoretical PI was 8.35.The phylogenetic analysis revealed that four EsPrxs genes can be subdivided into two groups,EsPrx,EsPrx3 and EsPrx4 belonged to the typical 2-Cys subgroup and the EsPrx5 belonged to the atypical 2-Cys subgroup.Multiple sequence alignment and homology analysis and revealed that all of the Prxs had two highly conserved Cys.The EsPrx3 and EsPrx4 sequences shows a consensus region FYPLDFTFVCPTE in the N-terminal and a consensus region GEVCPA in the C-terminal which are signatures of 2-Cys Prx.The EsPrx5 also have two signature motifs VPGAFTPGCSKTHLPG in the N-terminal and DGTGLTCSL in the C-terminal.The qRT-PCR showed that all these four genes were detected in hepatopancreas,gonads,gills,muscle,heart,intestine and hemocyte.The highest level of EsPrx,EsPrx3 and EsPrx4 were detected in hepatopancreas,and the highest level of EsPrx5 was detected in muscle.After challenged with the mRNA expression of EsPrx3,EsPrx4 and EsPrx5 all had significant rise in the hepatopancreas at 24 h post-challenge,values were 5,16.2,8.7 and 14.9-fold higher than those of the control group.At the same time,the activity of EsPrx also changed significantly,raised to 6.35 U/g.The coding regions of four genes were connected with vector pGEX-4T-1 then transformed into E.coli BL21(DE3).The recombinant proteins were successfully induced and purified.The antioxidant activity experiment results showed that recombinant proteins can reduce the H2O2.Also,different proteins showed different antioxidant abilities.The numerical value of EsPrx,EsPrx3,EsPrx4 and EsPrx5 recombinant proteins were 7.52,22.14,14.58 and 4.12 U/mgprot,respectively.All the results indicated that both EsPrx,EsPrx3,EsPrx4,and EsPrx5 genes could function as ROS scavenger,and might play important roles in immune response of pathogenic bacteria infection.