Expression Of Rice Cu/Zn-SOD May Limit The Synthesis Of GSH In Saccharomyces Cerevisiae

Aerobically growing organisms are unavoidably suffered from oxidative stress (ROS) and organisms has developed several mechanisms to remove these ROS.GSH and Superoxide dismutase(SOD) is the major antioxidant systems in the organisms. SOD is the enzyme that catalyzes the removal of superoxide radicals which are generated in various species. GSH can react with ROS and GSH was turned into GSSG. Some study has inplied there is some link between them, but no detail research results about it has been published till now.Saccharomyces cerevisiae was used as a model to study the functional link between SOD and GSH.Throgh the result of the SOD activity and GSHcontent determination ,a conclusion can be drawn that overexpression SOD in Saccharomyces cerevisiae can inhibit the GSH synthysis.In order to constitutive expression rice CuZn-SOD in Saccharomyces cerevisiae, the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) promoter from Saccharomyces cerevisiae was amplified by PCR with oligonucleotide primers and fused to the GFP gene.Strong green fluorescence from the cells of the Saccharomyces cerevisiae transformed with pGPD-GFP was detected by the fluorescence microscope. Using RT-PCR method, we got the rice CuZn-SOD cDNA and cloned in pGPD(drived from pYES2.0) to form pGPD-R-SOD.pGPD-R-SOD was transformed to the Saccharomyces cerevisiae to express rice CuZn-SOD. The total protein was extracted from the transformants and analyzed by SDS-PAGE .There is a obvious band about 16 KDa on the gel which was absent in the control cells.Using native gel and NBT method we can detect the activity of Cu-Zn-SOD .The activity of the SOD from the transformed Saccharomyces cerevisiae is about 50U/mg total protein,but the activity of the SOD from the wild type Saccharomyces cerevisiae is about 20U/mg total protein.From the results of the determination of SOD activity, we can conclude that the rice Cu/Zn-SOD gene can express in Saccharomyces cerevisiae efficiently.GSH content was determined by DTNB method. From the results of GSH content determination, the GSH content of the wild type yeast was almost the same as the yeast expressing GFP, but was higher than the yeast expressing rice Cu/Zn-SOD under heat shock and normal condition. In this study,we demonstrate that the heterologous expression of a rice Cu/Zn-SOD gene in the Saccharomyces cerevisiae may limit the synthesis of endogenous GSH under heat shock and normal condition.The results of this research show the functional link between GSH synthesis and SOD activity and the interaction mechanism of different antioxidant system in vivo.