Molecular Cloning,Expression And Enzymatic Activity Analyses Of Antioxidant Gene GPx7 And GPx8 From Chinese Black Sleeper

Glutathione peroxidase（GPx）is one of the most important enzymes in the antioxidant system.It plays a role in oxidant stress,scavenging hydrogen peroxide and protecting the biofilms from oxidative damage.The theory of early warning with fish as sensitive organisms has been widely concerned by scholars all over the world.Recently,fish specific genes have been used as biomarkers to detect and evaluate the degree of heavy metal pollution.Therefore,it is of great significance to study glutathione peroxidase gene of Chinese black sleeper for the development of aquaculture and marine ecology.The Chinese black sleeper（Bostrychus sinensis）,belongs to the family Eleotridae,Order Perciforms,is a kind of eurysalt-euryther-temperature fish with strong hypoxic tolerance,usually inhabiting in shallow brackish water area of warm water.As it has the effect of accelerating wound healing,it is considered to be one of the edible fish with very high medicinal value.Moreover,it has strong drought tolerance and is very suitable for live transportation,so it has become an important breeding specie in the southeast coastal area.In recent years,the Marine environment has been seriously polluted by industrial,agricultural and urban sewage.The entry of heavy metal pollutants into fish can induce oxidative stress and reduce the immunity of fish,resulting in frequent disease and death of mariculture species.The health development of mariculture is challenged.In this study,two genes,GPx7 and GPx8,which have not been widely studied,were selected to study heavy metal stress of Chinese black sleeper.The medial lethal concentration(LC₅₀)of Cd²⁺for 96 hours was 61.44 mg/L（the approximate value was 60 mg/L）determined by the acute poisoning test of Cd²⁺in our laboratory.Different concentration of Cd²⁺（7.5,15,30 mg/L）were set to simulate the stress effect of Cd²⁺in environment pollutants on Chinese black sleeper.The BsGPx7 and BsGPx8 sequence characteristics,the changes of BsGPx7 and BsGPx8 gene expression patterns and the prokaryotic expression conditions of BsGPx7 were studied by using molecular biology techniques.The main results are as follows:1.The gene sequence of BsGPx7 coding region was obtained by molecular cloning.Sequence analysis reveal that the amino sequence encodes a signal peptide,so BsGPx7 was considered to be a secretory protein.And it also encoded a typical GPx family domain,a Redoxin domain and two catalytic residues(Cys⁵⁶,Cys⁸⁵).Homology analysis show that BsGPx7 has high homology with species of gobiidae of perciform.The GPx family domain and catalytic residues are highly conserved in species evolution.RT-PCR results show that the BsGPx7 was found ubiquitously expressed in 10 examined organs of Chinese black sleeper,with predominant expression in liver.The expression of BsGPx7 gene in liver,head kidney,gill and skin were significantly upregulated at different times under different concentration of Cd²⁺（P<0.05）.2.The gene sequence of BsGPx8 coding region was obtained by molecular cloning.Sequence analysis reveal that BsGPx8 has no signal peptide and may be an intracellular protein,a transmembrane region at its N-terminal.Encoding a GPx family domain,a Redoxin domain and a catalytic residue(Cys¹⁰⁹).BsGPx8 has high homology with species of gobiidae of perciform.The GPx family domain and catalytic residue are highly conserved in species evolution.RT-PCR results show that BsGPx8 was found ubiquitously expressed in 10 examined organs of Chinese black sleeper,with predominant expression in liver.The expression of BsGPx8 gene was significant upregulated In liver,gill and sin under different concentrations of Cd²⁺（P<0.05）.3.In this study,the recombinant expression vector p ET-28b（+）of BsGPx7 was constructed,and the recombinant BsGPx7 protein was obtained by prokaryotic expression.The optimal expression condition is determined as E.coil BL21 cells,which was induced by1m M IPTG at 37℃for 4 h.under this condition,the solutional protein was purified and the r BsGPx7 with high purity was obtained.The enzyme activity of r BsGPx7 protein was determined under different temperature and p H condition.The results show that r BsGPx7had poor thermal stability and narrow range of p H resistance.The purified r BsGPx7 had optimal activity at 35°C and p H 8.In conclusion,this study suggests that BsGPx7 and BsGPx8 are involved in the progress of resistance to oxidative stress induced by Cd²⁺,and may play a certain role in antioxidant defense.