酵母菌Y12667 ERG11基因删除及验证研究

Deletion and Verification of the Correct Recombination of the ERG11 Gene in Yeast Y12667

采用同源重组的方法删除酵母菌Y12667内源ERG11基因,并用PCR、Western、细胞计数和GC-MS的方法分别在基因水平、蛋白水平、细胞水平和功能代谢水平进行验证.结果表明,本研究成功删除了酵母菌Y12667内源ERG11基因,该基因删除菌能稳定表达外源性白念珠菌ERG11基因的同源蛋白.因此,该基因删除菌可作为下一步靶酶蛋白功能研究的基因工程表达操作菌.

ERG11 gene of Saccharomyces cerevisiae was chosen as target gene to be disrupted. PCR-based gene deletion strategy was employed to knock out this gene. Targeting of the replacement cassettes at the genomic locus was verified by PCR analysis with the Zeocin gene as a probe. The inactive function of CYPS1 was observed through protein by Western blot and lanosterol composing by GC-GS. The result shows that, with the new deletion cassette, ERG11 gene has been disrupted successfully in Y12667.