酿酒酵母中PiggyBac转座子筛选系统的构建

PiggyBac Transposon Screening System in Budding Yeast

PiggyBac(PB)是来源于粉纹夜蛾(Trichoplusia ni)的TTAA型转座子。PB转座子系统可作为一种插入型突变工具,应用于多种真核细胞中。本研究在酿酒酵母(Saccharomyces cerevisiae)中构建了一个基于PB转座子的筛选系统。系统由插入ADE2基因编码框的PB转座子及由半乳糖启动子调控表达的PB转座酶(PBase)两部分组成。在PBase诱导下,PB转座子可以从原始位点无痕移除并随机插入新的基因位点。PB转座子在转座过程中保持单一拷贝数,在每个突变细胞中只有一个插入位点,并可快速检测突变基因。实验结果表明:所构建的PB转座子筛选系统可应用于酿酒酵母中的基因筛选。

The piggyBac(PB)transposon is a TTAA-specific transposon isolated from the cabbage looper moth,Trichoplusia ni.The PB transposon system works in various eukaryotic cells,and is used as a tool for insertional mutagenesis.We here developed the PB transposon screening system for in vivo mutagenesis in the budding yeast Saccharomyces cerevisiae.We established a system for PB-based mutagenesis consisting of a yeast strain containing a PB element,which was inserted in the ADE2 gene coding sequence,and expressing the PB transposase gene under a galactose-inducible promoter.The PB element was precisely excised from and re-inserted into the genome by PBtransposase expression in the strain.The PB element keeps one copy during transposition,and only one insertion site was found in each mutant cell.The DNA region containing the inserted PB elements can be easily identified.This study suggests our PB-based screening system is useful for isolating genes required for phenotypes of interest in budding yeast.