Expression And Function Analysis Of Keap1 Of Cristata Plicata

Microcystin(MCs)are a class of hazardous cyclic heptapeptide toxin that causes severe toxicity to the culture environment of Cristaria plicata.Keap1-Nrf2 signaling pathway is the key to protect cells from various environmental toxicants.Kelch-like ECH-associated protein 1(Keap1)is a BTB-Kelch-type substrate adaptor protein of the Cul3-dependent ubiquitin ligase complex.Keap1 facilitates the ubiquitination and degradation of Nrf2.Under oxidative stress,Keap1-Nrf2 signaling pathway regulates the downstream phase II detoxification/antioxidant enzymes to producing antioxidant responses.In this study,we analyzed the Keap1 characteristic and functional and the transcriptional regulation of the Nrf2-Keap1 signaling pathway under MCs stimulation,which will help to understand the immune response mechanism of molluscs.1.Two types of Keap1(Keap1a and Keap1b)gene fragments were originally analyzed from the transcriptome data of C.plicata hemocytes in our laboratory.The specific primers were designed and used RACE PCR to acquire complete cDNA of CpKeap1 a and CpKeap1 b.The full-length CpKeap1 a cDNA sequence was 2952 bp,and was consisted of a 5'-untranslated region(UTR)of 762 bp,a 3' UTR of 351 bp with a poly(A)tail and an ORF of 1839 bp.The ORF encoded 612 amino acids with the predicted protein molecular weight of 68.33 kDa and the theoretical isoelectric point(pI)of 6.80.The full-length CpKeap1 b cDNA sequence was 3710 bp,and was consisted of a 5' UTR of 87 bp,a 3' UTR of 1928 bp with a poly(A)tail and an ORF of 1695 bp.The ORF encoded 564 amino acids with the predicted protein molecular weight of 63.88 kDa and the theoretical isoelectric point(pI)of 5.28.The amino acid sequence analysis showed that the sequence contained BTB domain,IVR domain,DGR domain,and was conserved in human,mouse and zebrafish.The cysteine residues of Cys-273 and Cys-288 in the IVR domain of mouse Keap1 are required for the inhibition of Nrf2 activity,while CpKeap1 a contains the corresponding two cysteine residues.Two cysteine residues are not present in CpKeap1 b.2.The expression of CpKeap1 a and CpKeap1 b genes was detected in differenttissues of C.plicata by real-time fluorescent quantitative PCR.The results showed that CpKeap1 a and CpKeap1 b genes were expressed in hemocytes,mantle,muscle,gill and hepatopancreas,and the highest is in hepatopancreas,followed by muscle,the lowest is in mantle.The hepatopancreas is the main detoxification organ in shellfish,and the highest expression level in the hepatopancreas indicated that it was involved in hepatopancreas immunity.3.After stimulation with microcystins,the expression levels of CpKeap1 a and CpKeap1 b genes were detected in hemocytes and hepatopancreas.The results showed that the expression of CpKeap1 a and CpKeap1 b were down-regulated in hemocytes and hepatopancreas compared with saline group.CpKeap1 a was significantly down-regulated at 12 and 24 h in hemocytes,and significantly down-regulated at 48 h in hepatopancreas.CpKeap1 b was significantly down-regulated at 48 h in hemocytes,and significantly down-regulated at 12 and 48 h in hepatopancreas.The results indicated that CpKeap1 might be involved in the immune of C.plicata.The expression of CpKeap1 was reduced to leading to reducing the ubiquitinated degradation of CpNrf2.Thus it was playing an important role in immune regulation.4.The muscle of C.plicata was injected with effective interference reagent CpNrf2-siRNA 323 or CpKeap1-siRNA1117.After 24 h,C.plicata was injected with MCs.Comparasion with control groups,the expression levels of Mn-superoxide dismutase(CpMnSOD),Cu/Zn-superoxide dismutase(CpCu/ZnSOD),thioredoxin(CpTRX),peroxiredoxin(CpPrx),selenium-dependent glutathione peroxidase(CpSe-GPx),glutathione S-transferases(Cpsigma-GST)and Catalase(CpCAT)genes in hepatopancreas of C.plicata was up-regulation after MCs challenged.CpNrf2 knockdown reduced MCs-induced mRNA levels of these genes in hepatopancreas from Nrf2-siRNA-injected C.plicata.In contrast,CpKeap1 knockdown increased MCs-induced the mRNA levels of these genes in hepatopancreas from Keap1-siRNA-injected C.plicata.The results suggested that MCs could activate the Nrf2-Keap1 signaling pathway and could regulate the Nrf2-dependent downstream antioxidant/detoxification genes to attenuate the toxicity of MCs.5.The C-terminal conserved region of CpKeap1 a gene was transferred to the expression vector PET-32 a after BamH1 and XhoI1 double enzyme,and theprokaryotic expression plasmid of PET-32a-CpKeap1a-DC was successfully constructed.The recombinant plasmid was transferred into E.coli Rosetta.The result indicated that the recombinant protein was detected in the form of inclusion bodies by SDS-PAGE gel electrophoresis,and the concentration of protein was 0.314 mg/mL.