| 论文题目: | Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker |
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| 作者: | Song Panpan, Liu Sha, Guo Xuena, Bai Xuejing, He Xiuping*, Zhang Borun |
| 联系作者: | He Xiuping* |
| 刊物名称: | Anal Biochem |
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| 年份: | 2014 |
| 影响因子: | 2.749 |
| 论文下载: | 下载地址 |
| 摘要: | With increasing application of Hansenula polymorpha in fundamental research and biotechnology, much more genetic manipulations are required. However, these have been restricted for the finiteness of selectable markers. Here, MazF, a toxin protein from Escherichia coli, was investigated as a counter-selectable marker in H. polymorpha. The lethal effect of MazF on yeast cells suggested it a candidate for counter selection in H. polymorpha. Markerless or scarless gene deletion in H. polymorpha were conducted based on selectable markers cassette mazF-zeoR, in which zeocin resistance cassette and mazF expression cassette were used as positive and counter-selectable markers respectively. For markerless deletion, the target region can be replaced by CYC1TT via two-step homologous recombination. For scarless deletion, the innate upstream region (5'UP) of target genes rather than CYC1TT mediates homologous recombination to excise both selectable markers and 5' sequence of target genes. Moreover, scarless deletion can be accomplished by using short homologous arms for the effectiveness of mazF as counter-selectable marker. The applicability of the strategies in markerless or scarless deletion of PEP4, LEU2 and TRP1 indicates that this study provides easy, time-efficient and host-independent protocols for single or multiple genetic manipulations in H. polymorpha. |
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