【摘要】 利用PCR-mediated Technique对酿酒酵母W303-1A金属硫蛋白基因(cup1)进行敲除,获得一株铜离子敏感型酿酒酵母(Saccharomyces cerevisiae W303-1A cup1△),其对Cu2+抑制浓度由1.2 mmol/L降为0.08 mmol/L;以pYX212载体为基本构架,以S.cerevisiae W303-1A cup1△为宿主菌,构建了以cup1作为筛选标记的pYX212M表达系统,并成功表达了绿色荧光蛋白基因(gfp),从而实现了高灵敏度铜抗性酿酒酵母表达系统的构建与应用.本研究构建的高灵敏度铜抗性酿酒酵母表达系统不仅廉价、安全,而且丰富了酵母的表达系统,同时对微生物的铜抗性机理及生物修复功能的研究具有指导意义.更多还原

【Abstract】 This study was aimed to construct a high sensitivity expression system of copper-resistant Saccharomyces cerevisiae. With the PCR-mediated technique which allows single-step deletion of chromosomal gene to knock out the cup1 gene of S. cerevisiae W303-1A, the lowest inhibition concentration of Cu2+ was determined by gradient dilution. The construction of the expression system pYX212M used pYX212 as the basic frame, cup1 as the selective gene and the S. cerevisiae W303-1A cup1△ as the host. The pYX212MGFP was constructed, transforming S. cerevisiae W303-1A cup1△ to observe whether the strain displayed green fluorescence. A copper-sensitive S. cerevisiae W303-1A cup1△ was obtained and its lowest inhibition concentration of Cu2+ was 1.2 mmol/L compared with 0.08 mmol/L of S. cerevisiae W303-1A. The expression system pYX212M was achieved. The green fluorescence was seen under the microscope by transforming pYX212MGFP into the S. cerevisiae W303-1A cup1△, demonstrating the successful expression of gfp. The results suggested that the high sensitivity expression system of copper-resistant S. cerevisiae can be constructed. It not only enriches the expression system of S. cerevisiae, but also provides guidance in the research of the copper resistance mechanisms and the bioremediation function of the microorganism.更多还原