Cloning, Expression And Functional Analysis Of Two Heme Oxygenase Genes In Roughskin Sculpin(Trachidermus Fasciatus)

Roughskin sculpin, Trachidermus fasciatus Heckel (Scorpaeniformes:Cottidae), is a commercial fish with seawater-freshwater migratory habit. However, with the increase of environmental pressure and the influence of human activites,the survival of the habitat and spawning grounds have been severely disrupted.Consequently, Roughskin sculpin is listed as critically endangered in Category Ⅱ of the National Key Protected Wildlife List. Establishing nature reserves and aquaculture are crucial alternatives which is pursued to recover its population in the wild.In the process of breeding, disease is the serious problem which limits its development.To efficiently solve disease problems, Roughskin sculpin immune response should be studied thoroughly.Heme oxygenase,a microsomal enzyme, catalyzes the rate-limiting enzymatic step of heme degradation.HO,which plays roles in anti-inflammation, antioxidant and antiapoptosis,cleaves heme to form three biologically active products: biliverdin,carbon monoxide and free divalent iron.In the present study, two genes(TfHO-1and TfHO-2) are cloned from Roughskin sculpin using rapid amplification of cDNA ends (RACE) approaches. Quantitative real-time PCR (qRT-PCR) is employed to assess the mRNA expression of the two genes in some tissues and their temporal expression in important immune organs and brain of Roughskin sculpin post E. coli LPS challenge and heavy metal exposure. To elucidate their biological functions,they are expressed in E. coli BL21(DE3).The main results are as follows:The full length cDNA of TfHO-1is962bp with a816bp ORF, encoding a protein of272amino acids with the predicted molecular weight of32.12kD and isoelectric point (pI) of6.40. TfHO-1gene contains a heme oxygenase signature sequence (AA133-153)and a transmembrane domain sequence (AA248-270) at N-terminal by predicting gene functionality. No signal peptide is detected in the protein. The homology of HO-1between Roughskin sculpin and the other species submitted to GenBank is analyzed. The similarity is61.68%-76.84%between Roughskin sculpin and the other fishes, and it is42.81%-46.69%among Roughskin sculpin, mammals and the other species. qRT-PCR analysis reveals the presence of TfHO-1transcripts in all detected tissues. The expression of TfHO-1mRNA is up-regulated post heavy metal exposure and LPS challenge. The prokaryotic expression of HO-1is successfully constructed in E. coil BL21(DE3),and the E. coil BL21(DE3) with recombinant vector, pET-30a(+)/HO-1,is induced to express protein by IPTG. The result shows that a32kD protein is strongly expressed in E. coil BL21(DE3). The recombinant protein form into inclusion body with induction by IPTG at37°C for4h. The recombinant protein is purified by High-Affinity Ni-IDA Resin. The activity assays of recombinant TfHO-1protein shows that its specific activity is0.926nmol bilirubin/mg/h. Enzyme activity assay of TfHO-1which is incubated at the different pH and temperature shows that its optimum pH is7.4and optimum temperature is37°C.Full length cDNA of TfHO-2(1149bp) is cloned with the same method. The cDNA containes an ORF of822bp which encodes274amino acids with the predicted molecular weight of36.15kD and isoelectric point (pI) of5.79. A heme oxygenase signature sequence (AA132-154) and a transmembrane domain sequence (AA255-272) are predicted in TfHO-2gene. No signal peptide is detected in the protein. The alignment result indicates that the similarity is higher than60%between Roughskin sculpin and the other fishes, and it is aslo higher than40%among Roughskin sculpin, mammals and the other species. TfHO-2is broadly expressed in all the examined tissues. The expression of TfHO-2is up-regulated post heavy metal exposure but not change significantly after challenged with LPS.The prokaryotic expression of HO-2is successfully constructed in E. coil BL21(DE3), and the E. coil BL21(DE3) with recombinant vector, pET-30a(+)/HO-2, is induced to express peotein by IPTG. The result shows that a36kD protein is strongly expressed in E. coil BL21(DE3).The recombinant protein form into inclusion body with induction by IPTG at37°C for4h.The recombinant protein is purified by High-Affinity Ni-IDA Resin. The activity assays of recombinant TfHO-2protein shows that its specific activity is1.359nmol bilirubin/mg/h. Enzyme activity assay of TfHO-2which is incubated at the different pH and temperature shows that its optimum pH is7.4and optimum temperature is37°C.