酿酒酵母ADH3基因的敲除

Deletion of ADH3 gene from Saccharomyces cerevisiae

设计含有与酿酒酵母(Saccharomyces cerevisiae)编码乙醇脱氢酶Ⅲ的ADH3基因ORF两侧序列同源的长引物,以质粒pUG6为模板进行PCR构建带有Cre/loxP系统的敲除组件。转化酿酒酵母YS3(Saccharomyces cerevisiae),并将质粒pSH65转入阳性克隆子。半乳糖诱导表达Cre酶切除Kanr基因,在YPD培养基中连续传代培养丢失pSH65质粒,在原ORF处留下一个loxP位点,获得ADH3单倍体缺陷型菌株。利用同样的方法再次敲除双倍体的另一个等位基因。最终获得ADH3双倍体基因缺陷型突变株YS3-ADH3。

The ADH3 gene could encoded alcohol dehydrogenase in Saccharomyces cerevisiae. The gene disruption cassette produced by PCR using the same long oligonucleotides which comprised 45 5nucleotides that anneal to sites upstream or downstream of the genomic target sequence to be deleted. After transformed the linear disruption cassette with a Cre/ loxP-mediated marker into the cells of Saccharornyces cerevisiae YS3, selected transformants were checked by PCR for correct integration of the cassette and concurrent deletion of the chromosomal target sequence. Once correctly integrated into the genome, the select marker could be efficiently rescued by transforming the plasmid pSH65 and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure. The expression of the Cre recombinase resulted in the removal of the marker gene, leaving behind a single loxP site at the chromosomal locus to make the pSH65 plasmid lost by streaking cells onto YPD plates. At last, the diploid mutant YS3-ADH3 was generated.