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为研究过量表达的超氧化物歧化酶基因MnSOD对玉米抗逆性的作用,构建了小麦来源MnSOD基因的单子叶植物高效表达载体,用基因枪法转化优良玉米自交系胚性愈伤组织。经潮霉素梯度浓度培养基筛选,从阳性愈伤组织再生获得9个正常结实的植株。其中5株经PCR和Southern印迹检测表现为阳性,表明外源基因已整合到玉米基因组中。提取SOD酶液,非变性聚丙烯酰胺浓度梯度凝胶电泳分离,用H2O25mmol/L抑制FeSOD和Cu/ZnSOD活性,氯化硝基四氮唑蓝染色检测MnSOD酶活性。Southern印迹呈阳性的5个植株,MnSOD酶活性均高于未转基因的对照。甲基紫精氧化损伤处理后,用电解质渗漏率法测定阳性株系的叶片渗透液的电导率。结果表明,转基因株系的抗氧化损伤能力显著高于对照。
In order to study the effect of overexpression of MnSOD on maize stress tolerance, an efficient monocot expression vector was constructed for maize MnSOD gene and transformed into embryogenic callus of maize inbred lines by particle bombardment. After screening with hygromycin gradient concentration medium, nine normal plants were obtained from the regeneration of the positive callus. Five of them were positive by PCR and Southern blotting, indicating that foreign genes have been integrated into the maize genome. The SOD enzyme solution was extracted and the non-denaturing polyacrylamide concentration gradient gel electrophoresis was used. The activity of FeSOD and Cu / ZnSOD was inhibited by H2O25mmol / L, and the enzyme activity of MnSOD was detected by nitroblue tetrazolium blue staining. Five Southern blot positive plants showed MnSOD activity higher than that of the untransformed control. Methyl Violet oxidative damage treatment, the electrolyte leakage rate method was used to determine the conductivity of the positive leaflet permeate. The results showed that the anti-oxidative damage ability of transgenic lines was significantly higher than that of the control.