摘要
利用转座子突变技术构建毕赤酵母菌株突变库,并分离出能够在含雷帕霉素(10 ng/mL)培养基中生长的突变株mut375.通过热不对称交错PCR(TAIL-PCR)获取转座子侧翼序列,对转座子的插入位点进行定位.转座子插入PAS_Chr2-2_0375基因,破坏了其开放阅读框的完整性,将该基因命名为kFPR1.本研究利用同源重组敲除kFPR1基因使毕赤酵母产生了雷帕霉素抗性,回补该基因功能的菌株则不能在含雷帕霉素的培养基上生长.基于kFPR1基因的特性,建立了一种适用于毕赤酵母的无标记基因操作方法.以kFPR1作为反向筛选标记成功构建了表达EGFP的重组菌株并且回收了ZeoR筛选标记,为毕赤酵母的遗传操作提供了新的筛选工具.
The mutation library of Pichia pastoris strains was constructed by transposon mutation technology,and the mutant strain mut375,which could grow in the medium containing rapamycin(10 ng/mL)was isolated.The flanking sequence of the transposon was obtained by thermal asymmetric interleaved PCR(TAIL-PCR),then the insertion site of the transposon was located.The transposon inserted into the PAS_Chr2-2_0375 gene,destroying the integrity of its open reading frame,and the gene was named kFPR1.In this study,kFPR1 gene was knocked out to make Pichia pastoris resistant to rapamycin by homologous recombination.The strains that complement the function of this gene can not grow on the medium containing rapamycin.Based on the characteristics of the kFPR1 gene,a marker-free gene manipulation method suitable for Pichia pastoris was established.kFPR1 as the counter-selectable marker,a recombinant strain expressing EGFP was successfully constructed and the ZeoR selection marker was recovered,providing a new selection tool for genetic manipulation of Pichia pastoris.
作者
牛硕
朱梅君
魏子贡
NIU Shuo;ZHU Meijun;WEI Zigong(School of Life Sciences, Hubei University, Wuhan 430062, China)
出处
《湖北大学学报（自然科学版）》
CAS
2022年第1期24-30,共7页
Journal of Hubei University：Natural Science
基金
国家自然科学基金(31402036)资助。
