酿酒酵母 ERG6基因缺失突变株的构建

设计含有与ERG6基因两侧序列同源的长引物,以质粒pBlue-Kan为模板进行PCR扩增,构建含有Cre/lox P系统的酿酒酵母ERG6基因敲除组件.将基因敲除组件转化至酿酒酵母(Saccharomyce cerevisiae)AD1-8,通过同源重组的方式使目的基因缺失,获得为lox P-Kan-lox P序列组件所替换而产生Kanr的阳性克隆子.然后再将质粒pHis-Cre转入阳性克隆子表达Cre重组酶敲除筛选标记,成功获得酿酒酵母ERG6基因缺失的突变株,并命名为AD1-8-δ.

Construction of ERG6Gene Deletion Mutant of Saccharomyces cerevisiae

The gene disruption cassette was produced by PCR using the long primers which comprised upstream or downstream of the genomic target sequences of ERG6and two lox P sites.The linear disruption cassette with a selection marker in between the two lox P sites was transformed into the cells of Saccharomyces cerevisiae AD1-8.After homologous recombination the positive transformants were selected and checked by PCR to confirm the integration of the cassette and concurrent deletion of the chromosomal target sequence.Once correctly integrated into the genome,the select marker could be efficiently deleted by the expression of the Cre recombinase.Then we obtained the ERG6 gene deleted haploid Saccharomyces cerevisiae and named as AD1-8-δ.