三种酵母启动子在毕赤酵母中的功能比较

Functional Analysis of Three Promoters from Yeast in Pichia pastoris

启动子是基因表达调控的重要顺式元件,启动子功能的强弱对于目的基因的表达非常重要。为找到一个启动转录功能较好的启动子,以绿色荧光蛋白(GFP)为报告基因,在毕赤酵母中研究不同启动子对外源蛋白表达的影响。首先采用PCR扩增的方法克隆了酿酒酵母甘油合成关键酶3-磷酸甘油脱氢酶基因启动子pScgpd,借助于载体pPIC9K和pGAPZB,构建pPIC9K-gfp和pPIC9K-PG,pGAPZB-gfp和pGPDZB-gfp,将重组载体分别转入毕赤酵母GS115和毕赤酵母X33中,在不同渗透压条件下培养重组菌,通过GFP的表达情况比较启动子pScgpd与甲醇氧化酶启动子pAOX1、3-磷酸甘油醛脱氢酶启动子pGAP的功能。荧光显微镜观察结果显示,pScgpd表现为受高渗条件诱导,重组毕赤酵母P.pastoris GS115和P.pastoris X33均能产生稳定的荧光,且在一定范围内随着葡萄糖或NaCl浓度的增加,GFP表达强度也有所增加;但分别与对应宿主P.pastorisGS115、P.pastoris X33的启动子pAOX1和启动子pGAP相比,表达水平仍较弱。

The promoter is an important cis-element for regulation of gene expression,the strength of the promoter function is very important for the expression of target genes.In order to find a better promoter for transcriptional initiation,the experiment compared different promoters in the expression of foreign proteins in Pichia pastoris,using the green fluorescent protein（GFP） as a reporter gene.The promoter of glycerol-3-phosphate dehydrogenase gene（Scgpd） which is the key enzyme in Glycerol synthesis from Saccharomyces cerevisiae was cloned by PCR,and introduced it into pPIC9K and pGAPZB to construct pPIC9K-gfp and pPIC9K-PG,pPGAPZB-gfp and pGPDZB-gfp simultaneously.The recombinant plasmids were transformed into Pichia pastoris GS115 and Pichia pastoris X33 by electroporation.In the medium containing glucose or NaCl with different concentrations for culturing the recombinant strains,GFP was detected by fluorescent microscopy.By the expression of GFP,the fuction of pScgpd and methanol oxidase promoter pAOX1,3-GPDH promoter pGAP was compaired.Fluorescence microscopy showed pScgpd induced by the performance of hypertonic conditions.The gene gfp was functionally expressed under the control of the promoters in recombinant Pichia pastoris.Furthermore,the expression of the gene gfp at different level was conducted by the different concentrations of glucose or NaCl within a certain range for the recombinant strains.However,compared with the promoter pGAP and pAOX1 corresponding to the host P.pastoris GS115 and P.pastoris X33,the expression level under the control of the promoter pScgpd is still weak.