The Regulatory Function Of Nrf2/ARE Pathway On Prx5 In Cristaria Plicata

Cristaria plicata is the most vital freshwater mussel.It is susceptible to bacterial and microbial infections,which greatly influence on its pearl-breeding ability,and is very significant to explore its molecular immunity.Nrf2/ARE is a key pathway for cells to defend allogenic material or authigenic stimulation,reduce oxidative stress and mediate antioxidants.Peroxiredoxins（Prxs）,which is an enzyme in antioxidant defense system,and is one of the lower reaches target genes in Nrf2/ARE and is controlled by Nrf2/ARE.Prx5 can be localized in the mitochondria,peroxisome,tenuigenin and cytoplasm of mammalian cells.It not only protects cells from oxidative stress and apoptosis but also prolongs the lifespan of the cells.The enzyme also provides protection against mitochondrial or nuclear DNA damage.In addition,Prx5 is involved in the immunity reaction of multiple species after microbial infection.In this study,the c DNA of Prx5（named Cp Prx5）was cloned by RACE PCR.Clustalx software was used for similarity comparison with Cp Prx5.The c DNA sequence length of Prx5 was 1420 bp,and the 5’UTR and 3’UTR were 26 bp and 840bp,respectively.The 3’UTR had A poly A tail.The ORF was 567bp long and encodes189 amino acids containing two conserved cysteine residues（Cys78 and Cys179）.The molecular mass of the calculated protein was 20.69 KDa and the theoretical isoelectric point was 8.97.The amino acid sequence of Cp Prx5 was similar to Prx5 of other species（52.33-59.6%）,and the amino acid sequence contained a Pfam Ahp C＿TSA domain located at 35-159 AA.A phylogenetic tree was constructed to classify Prx5 as atypical2 cys Prx according to the number of conserved cysteine residues（cys）.Cp Prx5 was distributed in all tissues,but the highest expression was hepatopancreas,then gills,kidneys.LPS,PGN,Poly I:C stimulation resulted in up-regulated Cp Prx5 expression in hepatopancreas,gills and kidneys.DE3-PGX-4T-1-Cp Prx5 was showing resistance to H₂O₂.By constructing DTT/Fe³⁺/O₂ mixed functional oxidase system,hydroxyl radical attacked plasmid DNA,and the superhelix plasmid DNA changed from open loop state to a deletion state.With the increase of Cp Prx5 protein concentration,the upper helical form of plasmid DNA was protected.The expression of Cp Prx5 in hepatopancreas and gills was up-regulated after interference with keap1,and down-regulated after interference with Nrf2.The concentration of Cp Prx5 recombinant protein with GST label was 0.320 mg/m L after purification.A great deal of Cp Prx5 was localized in the293T nucleus,a small amount in cytoplasm,and Cp Maf K was localized in nucleus.GST-Pull down tested the ability of Cp Maf K and Cp Prx5 to bind in vitro.Gel block experiment results showed that Cp Maf K/Cp Nrf2 had a regulatory effect on the promoter of CpPrx5.