摘要
设计与酿酒酵母编码乙醛脱氢酶ALD4基因ORF两侧序列同源的删除组件引物,以质粒pUG6为模板进行PCR构建带有Kanr选择标记的删除组件。通过醋酸锂转化法将删除组件导入酿酒酵母菌,采用G418筛选阳性克隆子,将质粒pSH65转入阳性克隆子。半乳糖诱导pSH65表达Cre酶切除Kanr基因,在ALD4基因ORF处留下1个loxP位点,通过2次的转化筛选验证获得ALD4双倍体基因缺陷型突变株Y01-ALD4。
In order to disrupt the ALD4 gene coding aldehyde dehydrogenase in Saccharomyces cerevisiae, a pair of oligos, which were gene analogues to ambilateral sequence of ORF in ALD4, were synthesized for disruption. Using plasmid pUG6 as template for PCR amplification, the disruption cassette was constructed with Kanr as selective marker. The recombinant plasmid pSH65 was transformed into Saccharomyces cerevisiae by LiAc method and the positive clones were screened by G418. After the induction of galactose, the Cre enzyme was expressed to knock out Kanr gene and a loxP loci was formed near ALD4 ORE. A mutant named Y01-ALD4, with ALD4 amphiploid gene defect, was obtained after transformation, screening and validation by two times.
出处
《中国酿造》
CAS
北大核心
2009年第3期20-24,共5页
China Brewing
基金
国家高技术研究发展计划"863计划"(2007AA100701)
农业部"948"重大项目资助(2006-G37)
国家公益性行业(农业)科研专项(nyhyzx07-019)
关键词
酿酒酵母
乙醛脱氢酶
ALD4
基因删除
Saccharomyces cerevisiae
aldehyde dehydrogenase
ALD4
gene disruption
