摘要
热带假丝酵母是重要的工业生产菌株,但表达系统还不完善导致代谢工程育种手段受到限制。探索新的表达元件对热带假丝酵母的基因工程育种十分重要。本文通过多重序列比对从热带假丝酵母ATCC 20336基因组中扩增得到一个具有较高活性的磷酸甘油酸激酶基因(PGK1)启动子,以酵母增强型绿色荧光蛋白(yeGFP3)作为报告基因,整合到热带假丝酵母基因组上并对其表达活性进行研究。通过分离300、550、750 bp和846 bp4个长度的启动子片段分别表达yeGFP3,转化XZX宿主菌获得P-1、P-2、P-3和P-4共4个转化子。荧光显微镜结果表明,随着启动子长度的截短,荧光逐渐减弱,前3个转化子的相对荧光强度分别为P-4的4.98%、9.84%和48.4%。同时分析了4个转化子的yeGFP3转录水平,转录水平分别为P-4的5.6%、13.8%和60%。初步判断PGK1启动子至少存在2个上游激活序列(UAS)。
Candida tropicalis is an important industrial strain and can overproduce long chain dicarboxylic acid.However,genetic manipulations were still difficult because of lack of effective expression elements such as promoter and plasmid.Therefore,screening novel promoter is of great significance for metabolic engineering of C.tropicalis.In this study,phosphoglycerate kinase gene(PGK1)promoter was cloned from C.tropicalis ATCC 20336 and its function was investigated.The 846 bp PGK1 promoter was fused into a yeGFP3 reporter gene and transformed into C.tropicalis XZX host.The results showed that promoter could effectively regulate yeGFP3 expression.Then,a series of truncated promoters(including 300,550,750 bp)were cloned and used for expression of yeGFP3 reporter gene in C.tropicalis.Transformants harboring various integrative yeGFP3 reporter gene cassettes were obtained and confirmed.The relative fluorescence intensities of 300,550,750 bp promoters were 4.98%,9.84%and 48.4%,respectively,when using 846 bp PGK1 promoter as a control.Furthermore,the transcriptional levels of yeGFP3 in different transformants were detected by RT-qPCR and the results agreed to the fluorescence intensity evaluations.The results indicated that two upstream activation sequences presumably existed in the PGK1 promoter.In summary,PGK1 promoter would provide a fundamental expression element for engineering C.tropicalis in future.
作者
周静雨
陈献忠
张利华
沈微
樊游
ZHOU Jingyu;CHEN Xianzhong;ZHANG Lihua;SHEN Wei;FAN You(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2020年第3期95-103,共9页
Journal of Food Science and Biotechnology
基金
江苏省基础研究计划(自然科学基金)项目(BK20171138)。
关键词
热带假丝酵母
磷酸甘油酸激酶
启动子
绿色荧光蛋白
上游激活序列
Candida tropicalis
phosphoglycerate kinase
promoter
green fluorescent protein
upstream activating sequence
