利用荧光蛋白研究产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因CgGPD启动子

[目的]克隆产甘油假丝酵母(Candida glycerinogenes)胞浆3-磷酸甘油脱氢酶基因CgGPD的启动子(PCggpd),并通过报告基因gfp的差异表达来研究葡萄糖浓度对PCggpd在酿酒酵母(Saccharomyces cerevisiae)中的诱导特性.[方法]采用PCR扩增的方法分别从产甘油假丝酵母基因组和pCAMBIA1302载体中克隆出CgGPD的启动序列PCggpd和绿色荧光蛋白基因gfp.将两个基因同时构建到酿酒酵母表达载体pYX212-zeocin中,构建时将绿色荧光蛋白基因gfp置于CgGPD的启动序列下游,获得重组质粒pYX212-zeocin-PCggpd-gfp.通过电击转化酿酒酵母W303-lA.将重组酿酒酵母S.cerevisiae W303-1A-GFP置于不同葡萄糖浓度培养基中进行培养,利用荧光显微技术对其进行荧光检测.[结果]重组酿酒酵母能产生稳定的荧光,当葡萄糖浓度为2%时,重组酿酒酵母在YEPD培养基中产生较弱的荧光,随着葡萄糖浓度的升高,荧光强度有明显的增强.[结论]PCggpd属于环境胁迫诱导型启动子,高浓度的葡萄糖能诱导PCggpd启动绿色荧光蛋白的高水平表达,这对完善产甘油假丝酵母的遗传背景研究,阐明其高产甘油的机理具有重要意义.

Analysis of CgGPD gene promoter from Candida glycerinogenes by fluorescent protein

[Objective] We cloned the promoter of glycerol-3-phosphate dehydrogenase gene(CgGPD) from the Candida glycerinogenes, and studied its functional regulation under high osmotic stress condition. [Methods] We amplified the 950 bp promoter of CgGPD from C. glycerinogenes and the green fluorescent protein gene (gfp) from pCAMBIA1302 vector by PCR and introduced them into a modified vector pYX212-zeocin simultaneously. The recombinant plasmid pYX212-zeocin harboring both the promoter of CgGPD and gene gfp was transformed into S.cerevisiae W303-1A by electroporation. In the medium containing glucose with different concentrations for culturing the recombinant strain S. cerevisiae W303-1A--GFP the green fluorescence was detected by fluorescent microscopy. [Results] The gene gfp was functionally expressed under the control of the promoter of CgGPD in S. cerevisiae. Furthermore, the expression of the gene gfp at different level was conducted by the different osmotic stress for the recombinant strain. The green fluorescence was less intensive when the concentration of glucose was low for culturing the recombinant strain, but it became much more intensive when the concentration of glucose increased. [Conclusion] The promoter of CgGPD is an inducible promoter that can be induced significantly by the high concentration of glucose. The promoter will facilitate further studies on the mechanism of glycerol synthesis from C. glycerinogenes WL2002-5 under osmotic stress conditions.