Expression Of Cu,Zn-Superoxide Dismutase Gene From Kluveromces Lactis In Saccharomyces Cerevisiae

Superoxide dismutase (EC 1.15.1.1; SOD) is metal-containing enzymes that widely exist in organisms. According to their metal cofactor, three diferent classes of superoxide dismutases can be distinguished: Cu,Zn-SOD, Fe-SOD and Mn-SOD. Cu,Zn-SODs are predominantly found in the cytosol and peroxisomes of eukaryotes. They have also been detected in chloroplasts and in some bacterial strains. There are two kinds of copper- and zinc-superoxide dismutase: cytosolic Cu,Zn-SOD and extracellular SOD (EC-SOD). SOD catalyzes the dismutation of superoxide free radical and produces hydrogen peroxide and oxygen. It plays an important role in protection against oxidative stress, and the plasmalemma from the reactive oxygen species.In this work, we investigated the function of SOD in the yeast grown in poor environment. A 465bp DNA fragment of Cu,Zn-SOD structure gene from Kluveromces lactis and a 1214bp DNA fragment of GFP with PGK1 promoter and AT termianator from pUCm-T plasmid were amplified with PCR method. A recombinant plasmid YEplac195-PSGA was constructed where SOD gene and GFP gene were inserted into YEplac195. Through colony PCR and fluorescence screen, it was sure that the recombinant plasmid has expressed. Result indicated that stains harbroring YEplac195-PSGA produced more SOD than stains transformed with YEplac195-PGA when exposed to paraquat.SOD-GFP was then obtained from YEplac195-PSGA with SphI and EcoRI digestion. YCplac33-PSGA plasmid was constructed by inserting SOD-GFP gene into YCplac33. Result indicated that strains transformanted with YEplac195-PSGA produced more SOD, and the thermotolerance of strains harboring YEplac195-PSGA are higher than strains transformanted with YCplac33-PSGA.