| Roughskin sculpin Trachidermus fasciatus Heckel (Scorpaeniformes:Cottidae), is a traditional commercial fish. However, with the increasing environmental pressure, water pollution and the construction of water conservancy project, the habitats, breeding grounds and migration routes of roughskin sculpin have been affected or destroyed. With the sharp drop in the number of the species and the dramatically narrowed distribution, roughskin sculpin has been listed in the second order of protected aquatic animals in China. Aquaculture and the establishement of nature reserve have become an important strategy for recovering its population in the wild. However, in the process of breeding, disease is the serious problem. Therefore, research about immune defense, screening and cloning of associated genes of roughskin sculpin, may have important implications for the protection and breeding of roughskin sculpin.To further sthdy the immune system of roughskin sculpin, three kinds of detoxification and immune related glutathione S-transferase (GSTA, GSTO and MGST3) are selected. The detoxification process after xenobiotics enter the living body can be roughly divided into three phases. GSTs as an important member of the phase Ⅱ detoxifying enzymes, can catalyze GSH conjuncte with increased polarity toxic metabolites which are created from enzymes metabolism of phase Ⅰ. The conjunction can make polarity toxic metabolites more soluble or be low toxic metabolites, and be elimated them after a series of enzyme reactions of the phase Ⅲ. In the present study, three genes 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 three 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 TfGSTA is988bp with a672bp ORF, encoding a protein of223amino acids with the predicted molecular weight of25.5kD and isoelectric point (pI) of6.76. No signal peptide is detected in the protein. The homology of GSTA between roughskin sculpin and the other species submitted to GenBank is analyzed. The similarity is60.89%-87%between roughskin sculpin and the other fishes, and it is50.66%-54.63%among roughskin sculpin, birds and mammals. qRT-PCR analysis reveals the presence of TfTrxl transcripts in all detected tissues. The expression of TfGSTA mRNA is up-regulated post LPS challenge and heavy metal exposure. The prokaryotic expression of GSTA is successfully constructed in E. coil BL21(DE3), and the E. coil BL21(DE3) with recombinant vector, pET-30a(+)/GSTA, is induced to express peotein by IPTG. The result shows that a26kD protein is strongly expressed in E. coil BL21(DE3). The recombinant protein form into inclusion body with induction by IPTG at37℃for4h. The recombinant protein is purified by High-Affinity Ni-IDA Resin. The glutathione S-transferase activity assays of recombinant TfGSTA protein show that its specific activity is9.649±0.092μmol/mg/min. Enzyme activity assay of TfGSTA which is incubated at the different pH and temperature show that its optimum pH is9.0and its optimum temperature is25℃. With the increase of urea or SDS concentration, the activity of the recombinant protein TfGSTA gradually reduce.Complete TfGSTO cDNA sequence1136bp in length is cloned. It contains an ORF of720bp which encodes239amino acids. The alignment result indicates that the similarity is70.71%-77.41%between roughskin sculpin and the other fishes, and it is55.79%-56.61%among roughskin sculpin, birds and mammals. TfGSTO is broadly expressed in all the examined tissues. The expression of TfGSTO is up-regulated post LPS challenge and heavy metal exposure. The prokaryotic expression of GSTO is successfully constructed in E. coil BL21(DE3), and the E. coil BL21(DE3) with recombinant vector, pET-30a(+)/GSTO, is induced to express peotein by IPTG. The result shows that a33kD protein is strongly expressed in E. coil BL21(DE3). Most of the recombinant protein form into inclusion body with induction by IPTG at37℃for4h, while partial of it is soluble with induction by IPTG at25 ℃overnight. The recombinant protein is purified by High-Affinity Ni-IDA Resin. The glutathione S-transferase activity assays of recombinant TfGSTO protein show that its specific activity is6.260±0.052μmol/mg/min. Enzyme activity assay of TfGSTO which is incubated at the different pH and temperature show that its optimum pH is9.0and its optimum temperature is37℃. With the increase of urea or SDS concentration, the activity of the recombinant protein TfGSTO gradually reduce.Full length cDNA of TfMGST3(1076bp) is cloned with the same method. The cDNA containes an ORP of465bp which encodes154amino acids with the predicted molecular weight of16.81kD and isoelectric point (pI) of9.39. The similarity of amino-acid sequence is69.74%-76.13%between roughskin sculpin and the other fishes submitted to GenBank, and it is57.79%-62.34%among roughskin sculpin and amphibia, birds and mammals. TfMGST3ubiquitously express in all the detected tissues. In the brain and main immune organs, the expression of TfMGST3is significantly induced post LPS chanllege and heavy metal exposure. The recombinant vector, pET-30a(+)/MGST3, is constructed, then transformed into E. coil BL21(DE3), and is induced to express protein by IPTG. The protein product is identified by SDS-PAGE. The result shows that a22kD protein is weakly expressed in E. coil BL21(DE3). But, there is little effect on the percentage of target protein with the treatment of different concentration of IPTG, temperatures and time. The glutathione peroxidase activity assays of recombinant TfMGST3protein show that its specific activity is2.073±0.326μmol/mg/min. These findings suggest that TfMGST3probably play an essential role in detoxification and antioxidant defense in physiological state. |
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