酿酒酵母NST1基因敲除菌的构建及其对抗氧化的影响  

Effects of NST1 gene knockout in Saccharomyces cerevisiae on oxidation resistance

作　　者：朱红 王梦飞 焦宏亮 王金鹏 周华[1] 蔡恒[1] ZHU Hong;WANG Mengfei;JIAO Hongliang;WANG Jinpeng;ZHOU Hua;CAI Heng(College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211800,China)

机构地区：[1]南京工业大学生物与制药工程学院,江苏南京211800

出　　处：《生物加工过程》2020年第5期549-555,共7页Chinese Journal of Bioprocess Engineering

基　　金：国家重点基础研究发展计划(973计划)(2013CB733904);江苏省研究生科研与实践创新计划(KYCX18_1116)。

摘　　要：构建一株酿酒酵母(Saccharomyces cerevisiae)NST1基因缺失菌株并研究其对抗氧能力的影响。以酿酒酵母BY4741中的NST1基因为研究对象,利用Cre-LoxP基因敲除系统将NST1基因与G418抗性基因(kan^r)相替换,实现目的基因的敲除。通过稀释点样实验、荧光电子显微镜检测和荧光定量PCR等技术分析NST1基因在酿酒酵母抗氧化体系中的作用。经过G418抗性筛选和基因组PCR鉴定,成功获得了NST1基因缺失菌株nst1Δ,实验数据显示nst1Δ重组菌胞内ROS增多,并且降低了细胞壁完整性信号通路(CWI)途径下游基因RLM1的表达水平,表明NST1基因的敲除对酿酒酵母BY4741的抗氧化性能有影响。We constructed a Saccharomyces cerevisiae mutant strain with gene NST1 gene knockout to study the effects of NST1 gene on oxidation resistance.With the gene NST1 in Saccharomyces cerevisiae BY4741 as research object,the gene NST1 fragment was replaced with G418 resistance gene(kan^r)by using Cre-LoxP knockout technique,and then the target gene was knocked out successfully.The function of NST1 in oxidation resistance of yeast was analyzed by experiments of dilution point sample experiment,fluorescence electron microscope,fluorescence quantitative PCR analysis.The results show that a gene NST1 deletion strain(named nst1Δ)was obtained successfully by G418 resistance screening and genome PCR identification.The intracellular reactive oxygen species(ROS)of nst1Δrecombinant increased and the expression level of the downstream genes RLM1 decreased in the cell wall integrity(CWI)pathway.Knockout the NST1 gene has a great influence on the antioxidant properties of yeast BY4741.

关 键 词：酿酒酵母 基因敲除 NST1 抗氧化 

分 类 号：Q78[生物学—分子生物学]