基于Egfp启动子高通量筛选方法的研究

本研究以载体pUC-19为基础质粒，引入编码增强型绿色荧光蛋白的Egfp为报告基因，选用PGK为启动子，构建重组质粒pUC-PEBBK。通过PCR扩增出重组盒BATs-PGKp-Egfp-PGKt-KanMX-BATx，将其与酿酒酵母AY15单倍体α5同源重组，获得重组菌株α5-PEBBK。使用荧光显微镜和酶标仪检测EGFP在酿酒酵母中的表达，检测到重组菌荧光强度（RFU）为523，亲本菌株为53，是原菌的10倍，证明Egfp在PGK启动子调控下在酿酒酵母α5中能够正确表达。通过培养基优化，排除了培养基成分酵母浸粉和蛋白胨对绿色荧光蛋白检测的干扰，确定了利于Egfp表达和快速检测的筛选培养基BSM1。考察了接种量和培养时间对Egfp荧光表达和菌体生长的影响，确定了最佳48孔板培养条件为接种量40 μL，培养时间为36 h。高效、灵敏的高通量筛选方法的建立为后续系列表达强度启动子的筛选奠定了良好的基础。
The recombined vector pUC-PEBBK was constructed based on the pUC-19 as the base plasmid, which seclected PGK as the promoter and Egfp of the encodingenhanced green fluorescent protein as the reporter gene in this study. The recombinant cassette BATs-PGKp-Egfp-PGKt-KanMX-BATx was amplified by PCR,which was homologous recombination with Saccharomyces cerevisiae haploid AY15-α5 in order to construct the haploid recombinant α5-PEBBK. The expression of Egfp in Saccharomyces cerevisiae was detected using fluorescence microplateand fluorescence microplate. It was showed that the recombinant RFU of Egfp in the haploid recombinant α5-PEBBK was 523, which was 10.0 fold higher than the parental strain with RFU 53. It was concluded that the Egfp could be correctly expressed in Saccharomyces cerevisiae AY15-α5 under the control of PGK promoter. BSM1 was selected as the optimal medium for reducing the interference on fluorescence signal by the optimization of medium. The effects of inoculation quantity and time on Egfp fluorescence expression and microorganism growth were investigated. The optimal culture conditions were 40 μL of inoculation and 36 h of incubation time. The establishment of high efficient and sensitive high-throughput screening method provides a good foundation for the selection of subsequent series of expression intensity promoters.