Optimization of Real-Time Polymerase Chain Reaction Conditions of Some Superoxide Dismutase Genes for Analysis in Wheat

Abstract

Environmental stress causes a significant decrease in crop quality and losses of productivity every year. One of the important mechanisms by which plants are damaged in adverse environmental conditions is the overproduction of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals. In addition, such oxidative stresses have been shown to occur in plants exposed to high and low temperatures, particularly to high light intensities, drought, the presence of air pollutants such as ozone or sulfur dioxide, ultraviolet light, and herbicides. Hydroxyl radicals instantly react with proteins, lipids, and deoxyribonucleic acid, causing rapid cell damage. Therefore, plants have developed enzymatic and nonenzymatic mechanisms that efficiently scavenge oxygen radicals. However, hydroxyl radicals are too reactive to be eliminated enzymatically, so the formation of radicals is limited by the release of O2 and H2O2. Superoxide dismutases, which are also the subject of this study, are key enzymes that scavenges superoxide radicals (2O2 + 2H+ ? H2O2 +O2), contain metals, and act as the first line of antioxidant defense. In this study, eight superoxide dismutase genes were chosen for real-time polymerase chain reaction optimization. Specific primers were designed, and annealing temperature optimization was performed for realtime polymerase chain reaction analysis using deoxyribonucleic acid from wheat sample. In addition, annealing temperature optimization for ?-actin specific primers were performed using the same deoxyribonucleic acid sample. ?-actin is a housekeeping gene with a constant expression profile that is commonly used as a normalizing factor in expression profile studies. Evaluation of the relative and absolute values of superoxide dismutase gene expressions and the changes of superoxide dismutase gene expressions over time and under different conditions can be easily studied using the established real-time polymerase chain reaction protocols. These studies can provide important information on wheat coping mechanisms under different stress conditions.