摘要
The expression of a rice Cu/Zn superoxide dismutase （Cu/Zn-SOD） in Saccharomyces cerevisiae regulated by GAPDH promoter, involved in the inhibition of endogenous Glutathione （GSH） synthesis, and the competitive expression was detected by constructing the expression vector transferred Cu/Zn-SOD gene into wild-type S. cerevisiae. Transcription and expression of the Cu/Zn-SOD gene in S. cerevisiawere were confirmed by northern blot and SDS-PAGE, respectively, and activity of the Cu/Zn-SOD from crude extracts was enzymatically detected based on the effect of nitroblue tetrazolium （NBT） after running a native polyacrylamide gel. The GSH synthesis was also tested by DTNB （5, 5＇-Dithiobis （2-nitrobenzoic acid）） method. Results showed that GSH synthesis was evidently suppressed by the expression of Cu/Zn-SOD gene in both control and heat shock strains. It implied that the expression of the Cu/Zn-SOD gene in S. cerevisiae has more potential facility in response to oxidative exposure than that of endogenous GSH, although Cu/Zn-SOD and GSH were both contributed to the function of oxygen radical oxidoreduction.
本研究验证了水稻铜锌超氧化物歧化酶的表达对酿酒酵母本身谷胱甘肽合成的竞争性抑制作用。构建的转基因酵母表达载体主要是将水稻铜锌超氧化物歧化酶基因转入野生型的酿酒酵母中,该基因的转录和表达分别通过NORTHERNBLOT和变性聚丙烯酰胺凝胶进行确认,初提物活性通过非变性聚丙烯酰胺凝胶上NBT酶活染色的方法进行检测。此外,谷胱甘肽的含量通过DTNB染色法标定,再用标准曲线核算。结果表明,对照和热激的菌落中谷胱甘肽的合成都明显受到水稻铜锌超氧化物歧化酶的表达的抑制。尽管水稻铜锌超氧化物歧化酶和谷胱甘肽都具有消除活性氧自由基的作用,我们的结果表明,抗氧化胁迫反应中水稻铜锌超氧化物歧化酶比谷胱甘肽更具有潜在的优势。图3参18。
基金
overseas returned scholars’ starting working grants to AN Zhi-gang (Northeast Forestry University)
