现代食品科技2018，Vol.34Issue(1)：134-140,7.DOI:10.13982/j.mfst.1673-9078.2018.1.021
基于Egfp启动子高通量筛选方法的研究
High Throughput Screening of Mutant PGK Promoter Based on Egfp
摘要
Abstract
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.关键词
酿酒酵母/Egfp/PGK启动子/高通量筛选Key words
Saccharomyces cerevisiae/Egfp/PGK promoter/fluorescence detection引用本文复制引用
徐佳,皮莉,张玉,崔丹瑶,张翠英,肖冬光..基于Egfp启动子高通量筛选方法的研究[J].现代食品科技,2018,34(1):134-140,7.基金项目
国家自然科学基金项目(31471724) （31471724）
天津市科委自然基金项目(14JCZDJC32900) （14JCZDJC32900）
