Expression And Function Analysis Of Nrf2 And MafK Gene Of Cristata Plicata

Nrf2-ARE signaling pathway is an important way to regulate transcription expression of various antioxidant enzymes and cells against exogenous stimuli.At present,there are studies in mammals showing that many chemical substances can activate Nrf2 to initiate the regulatory expression of target genes and thus resist ROS and oxidative damage.Cristaria plicata is a kind of freshwater cultured oysters.In recent years,the frequency of disease has seriously affected its ability to raise the pearl.The study of the Nrf2-ARE pathway will help understand the immune response mechanism of shellfish and provide a theoretical basis for the healthy culture of Cristaria plicata.In this study,the cDNAs of Nrf2 and MafK genes of C.plicata were successfully cloned by transcriptome libraries and RACE PCR.The full-length Nrf2 gene is 4259 bp,consisting of a 482 bp 5?UTR,a 1113 bp 3? UTR,and a longest887 amino acid 2664 bp ORF.The predicted molecular weight is 99.94 kDa and the is PI is 5.09.Amino acid sequence analysis showed that Nrf2 had the highest homology with Mizuhopecten yessoensis(46%).With human,mouse,zebrafish,etc.have a conserved binding region(RRRGKNKVAAHNCRKR)and a conserved leucine zipper residue 802(L,leucine),813(R,arginine),823 Position(L,Leucine),Position 839(R,Arginine),Position 853(L,Leucine)and Position 863(L,Leucine).MafK is 2170 bp in length and consists of a 467 bp 5?UTR,a 1133 bp 3?UTR,and a 570 bp ORF that encodes 289 amino acids.The predicted protein molecular weight is 21.91 kDa and the PI is 9.56.CpMafK contains four highly conserved motifs(SUMO consensus motif,extended homologous region,basic region and leucine zipper leucine residue)and four highly conserved residues(Lys17,Arg77,Asn82 andTyr85)).Phylogenetic analysis showed that CpMafK was clustered with invertebrates and was most conserved with Crassostrea gigas.Quantitative real-time fluorescent detection of Nrf2 and MafK gene expression in different tissues of Cristaria plicata and changes in hepatopancreas and hemolymph after stimulation with microcystins.The results showed that the Nrf2 and MafK mRNAs were expressed in the mantle,the adductor muscle,the iliac crest,the hemolymph,and the hepatopancreas of pleated crested gills,and were all expressed in hepatopancreas.After stimulated by microcystins,the Nrf2 and MafK mRNAs in the experimental group and the control group all showed an upward trend.Nrf2 was upregulated to the highest level in the hemolymph tissue after 6 hours of stimulation,and stimulated in the hepato-pancreatic tissues.After h reached the highest.In the haemolymph group,the expression of MafK was significantly increased and reached the highest level after 3 hours of stimulation.In the hepatopancreas,it reached the highest level after 12 hours of stimulation but was lower than that of saline after 24 hours and 48 hours.The prokaryotic expression results showed that both pET-32-Nrf2-C and pET-32-MafK fusion proteins existed in the form of inclusion bodies,and after renaturation,active proteins were obtained.Nrf2-C and MafK polyclonal antibodies were obtained.The Gene Walking technology was used to clone the promoter sequence.The gel retardation assay showed that the CpMafK protein could bind to the sigma-GST promoter with high affinity in vitro.