Molecular Cloning And Expression Of 1-Cys And 2-Cys Peroxiredoxins From Cristaria Plicata

Cristaria plicata is one of the major "Freshwater pearl mussel" in China. With the development of in intensive culture and environmental deterioration, various diseases caused pathogens and chemical in C. plicata populations.The peroxiredoxins are a newly-defined family of antioxidants that maintain the balance of intracellular redox by reducing and detoxifying hydrogen peroxide and alkyl hydroperoxides in cells. In this study, a based study for 1-Cys Prx and 2-Cys Prx genes is to clarify two types of antioxidant proteins's role in the C. plicata immune system. Better understanding of the two types of antioxidant proteins will be beneficial to provide a theoretical basis for disease resistance and the development of health managementl in freshwater mussel aquaculture.1. Molecular cloning and expression of 1-Cys Prx from Cristaria plicataThe full length cDNA of CpPrx6 was of 1617 bp, contains a 5'-untranslated region (UTR) of 71 bp, a 3'UTR of 889 bp, and an open reading frame of 657 bp encoding 218 amino acids. The predicted molecular mass and estimated isoelectric point point (pI) of CpPrx6 were 24.24 kDa and 6.33, respectively. The conserved peroxidase catalytic center "PVCTTE" and a catalytic center for PLA2 activity "GKSW" were observed in the sequence, this indicated that it was a member of 1-Cys Prx. The secondary structures of CpPrx6 were constituted by 6α-helix and 12β-sheet. Blast and phylogenetic analysis showed that the amino acids sequences of CpPrx6 have highest similarity with 1-Cys Prx of Hyriopsis cumingii,Crassostrea gigas,Haliotis discus discus.Expression of CpPrx6 in different tissues of C. plicate was determined by RT-PCR. The result showed that the expression of CpPrx6 was detected in hemocytes, mantel, conductor muscle, hepatopancreas and gill. The gene expression in gill was significantly higher than other organizations. At 12h after injection by Aeromonas hydrophila, the expression levels of CpPrx6 in hepatopancreas increased. At 24h after injection, the expression levels resumed to normal levels. At 6h after injection, the experimental group and control group have obvious changes. At 12h, there was significant difference between these two groups.CpPrx6 was subcloned into the plasmid pET-32a(+) and the recombinant protein was expressed in Escherichia coli BL21(DE3). The experimental group appears a significantly 40 KDa a protin band than the control with SDS-PAGE after induced by IPTG which shows CpPrx6 can expressed in pET-32a(+) prokaryotic expression system.2. Molecular cloning and expression of 2-Cys Prx from Cristaria plicataThe full length cDAN of Cristaria plicata TPx (CpTPx) was of 1247 bp, contains a 5'-UTR of 90 bp, and a 3'-UTR of 566 bp. An open reading frame of of 591 bp encoding 196 amino acids. The predicted molecular mass and estimated isoelectric point(pI) of CpTPx were 21.8 kDa and 5.95. CpTPx amino acid sequences exhibited two highly conserved 2-Cys Prx signature domains named as F-motif "FYPLDFTFVCPTEI" and hydrophobic "VCPAGW". Especially, CpTPx also contain atypical 2-Cys Prx signature sequences "GGLG". Therefore, CpTPx was found to belong to the atypicla 2-Cys Prx subgroup..The secondary structure of CpPrx6 was constituted by 5α-helix and 7β-sheet with 2 random coil. Phylogenetic analysis CpPrx6 was clustered separately into Prxl subgroup.RT-PCR showed constitutive mRNA expression in in hemocytes, mantel, conductor muscle, hepatopancreas and gill. Expression level in gill and hepatopancreas were higher than other organizations. At 24 h after injection by Aeromonas hydrophila, the expression levels of CpTPx in hepatopancreas increased, At 6 h and 24 h after injection, the experimental group and control group have obvious changes. At 12 h, there was significant difference between these two groups.CpTPx was subcloned into the plasmid pET-32a(+) and the recombinant protein was expressed in E. coli BL21(DE3). The experimental group appears a significantly 37 KD a protein band than the control with SDS-PAGE after induced by IPTG wich showed CpTPx can be expressed in pET-32a(+) prokaryctic expression system. E. coli cells transformed with CpTPx coding sequences in pET-32a(+) showed resistance to H2O2 at 0-0.8 mM concentration by in vivo H2O2 tolerance assay.