热带假丝酵母磷酸甘油酸激酶基因启动子功能鉴定

Characterization of Phosphoglycerate Kinase Promoter fromCandida tropicalis

DOI：10.3969/j.issn.1673-1689.2020.03.013

中文关键词: 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

英文关键词: Candida tropicalis,phosphoglycerate kinase,promoter,green fluorescent protein,upstream activating sequence

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中文摘要:

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）。

英文摘要:

Candida tropicalisis 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 ofC. tropicalis. In this study，phosphoglycerate kinase gene (PGK1) promoter was cloned fromC. tropicalisATCC 20336 and its function was investigated. The 846 bpPGK1promoter was fused into a yeGFP3 reporter gene and transformed into C. tropicalis XZX host. The results showed that promoter could effectively regulateyeGFP3expression. Then，a series of truncated promoters (including 300，550，750 bp) were cloned and used for expression ofyeGFP3reporter gene in C. tropicalis. Transformants harboring various integrativeyeGFP3reporter 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 bpPGK1promoter as a control. Furthermore，the transcriptional levels ofyeGFP3in 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 thePGK1promoter. In summary，PGK1promoter would provide a fundamental expression element for engineeringC. tropicalisin future.

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