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Cloning of yeast suc2 gene and its targeted disruption in yeast genome
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FORTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 12
- DOI:
- 10.3321/j.issn:1000-2790.2000.12.005
- Key Word:
- 蔗糖转换酶;同源重组;(V)突变;(V)信号肽
Abstract： 目的克隆酿酒酵母的蔗糖转换酶基因(suc2),并定位突变酵母基因组中的suc2基因以获得无蔗糖转换酶表达的酵母品系.方法用P CR法从酵母Y190基因组中扩增出suc2 的成熟肽基因片段,克隆后测序.将色氨酸合成酶(TRP)基因片段插入suc2基因中, 构建成用于同源重组的suc2基因定位突变载体.导入酵母Y190,用营养缺陷筛选出suc2 基因突变的克隆.用PCR扩增并克隆突变后的suc2基因,用序列分析确定同源重组的发生.结果用 PCR从酵母基因组DNA中扩增出大小约1.5 kb的suc2基因片段,序列测定表明除585位有一点突变(AAC变为AAT)外,所得suc2基因与文献报道相同;构建的定位突变载体导入酵母细胞后 ,得到4株营养型符合的突变体;PCR表明这些突变体中均有suc2基因的同源重组;取其中1株的PCR产物进行序列测定证实了同源重组的发生.结论克隆了正确的编码蔗糖转换酶成熟肽的suc2基因片段;用同源重组方法在酵母Y190的基因组中定位突变了suc2基因 .
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