suc2基因的克隆及在酵母基因组中的定位突变  

Cloning of yeast suc2 gene and its targeted disruption in yeast genome

作　　者：孙强[1] 王冀姝[1] 陈萍[1] 柳天平 牛利国[1] 韩骅[1] 苏成芝[1] 

机构地区：[1]第四军医大学基础部生化与分子生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2000年第12期1447-1450,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金资助项目! (3 9970 3 76)

摘　　要：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 PCR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 5 85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AIM To obtain a yeast strain which does not secrete invertase by cloning suc2 gene from saccharomyces cerevisiae and disrupting it in the yeast genome. METHODS suc2 gene fragment which encoded mature invertase peptide was amplified by PCR using genomic DNA from yeast strain Y190 as a template, followed by cloning and DNA sequencing. A homologous recombination vector was constructed,in which suc2 gene was disrupted by deletion and insertion of selection marker TRP. The vector was used to transform yeast strain Y190 and the suc2-deficient yeast clones were selected using their auxotrophy. Homologous recombination was confirmed by amplifying mutated suc2 gene and DNA sequencing. RESULTS suc2 gene fragment of about 1.5 kb was amplified and cloned. DNA sequencing confirmed that, compared with the published sequence, the cloned suc2 gene had correct sequence except for one point mutation, but that did not alter the amino acid sequence of the encoded peptide. By transformation of yeast cells with a constructed gene disruption vector, four yeast clones were obtained which showed correct auxotrophy. PCR and DNA sequencing indicated that these yeast clones had the expected genotype. CONCLUSION suc2 gene is successfully cloned from yeast genome and a yeast strain that does not secrete invertase is established by homologous recombination.

关 键 词：蔗糖转换酶 同源重组 suc2基因 克隆 定位突变 

分 类 号：Q785[生物学—分子生物学] Q754