新型酵母蛋白表位标记和基因敲除质粒系统的构建及可行性验证

【目的】构建一套用于酿酒酵母基因功能研究的质粒。该套质粒结合pUG系列和pFA6a系列的优点,同时采用同尾酶实现蛋白表位标签的串联插入。【方法】利用PCR技术分别克隆pUG系列质粒的lox P位点、pFA6a质粒多酶切位点和ADH1终止子模块;通过重组连接各片段,构建pCLHN-TRP和pCLHN-URA质粒。在此基础上利用同尾酶实现多种蛋白表位标签的单个或串联重复插入,获得一系列蛋白表位标记质粒。最后,以ATG1、COX4和NHX1为例验证本质粒系列的性能。【结果】在本项工作中,我们共构建2种基因敲除用质粒和17种表位标记用质粒(涵盖1-8 FLAG、1-12 V5、3-9 HA、2-8MYC、GFP和m Cherry)。在几个靶基因上的应用证实了本套质粒的实用性。尤其值得指出的是,通过组合采用不同重复度的串联表位标签,在同一张膜上同时检测表达差异极大的不同蛋白而不使高表达蛋白信号饱和成为可能。【结论】本文所构建的pCLHN质粒系列是对现有酵母质粒工具的有益补充。

Construction of a novel plasmid system for epitope tagging and gene deletion in Saccharomyces cerevisiae

Objective] For yeast gene functional studies, we constructed a plasmid set combining the advantages of pUG and pFA6a plasmid series, and allowing convenient insertion of tandem epitope tags using isocaudomers. Methods] We cloned the loxP locus of pUG plasmids, the multiple restriction site of pFA6a plasmids and ADH1 terminator cassette by PCR. Through homologous recombination of the DNA fragments, we obtained plasmid pCLHN-TRP and pCLHN-URA. Using introduced isocaudomer sites, we then constructed additional tagging plasmids containing either single or multiple tandem copies of various epitope tags. Finally, we tested the performance of our plasmids using ATG1, COX4 and NHX1 as target genes. Results] We constructed two plasmids for gene deletion purposes, and seventeen plasmids for epitope tagging purposes (covering 1-8 FLAG, 1-12 V5, 3-9 HA, 2-8 MYC, GFP and mCherry). Testing on selected target genes demonstrated the usability of these plasmids. In particular, by combining different copy numbers of tandem epitopes, it was feasible to properly detect proteins with drastically different expression levels on the same blot without saturating the signal of high expression targets. Conclusion] The pCLHN plasmids constitute a beneficial complement to existing yeast plasmid tools.