酿酒酵母无标记转化子两种筛选方法比较  

Comparison of Two Selection Method of Saccharomyces cerevisia Transformants with No Marker

作　　者：徐民俊[1] 田小群[2] 付京花[3] 周世宁[1] 

机构地区：[1]中山大学生命科学学院有害生物控制与资源利用国家重点实验室,广东广州510275 [2]广州珠江啤酒股份有限公司,广东广州510308 [3]华南农业大学动科院,广东广州510642

出　　处：《酿酒科技》2008年第5期17-20,共4页Liquor-Making Science & Technology

基　　金：广东省自然科学基金项目(批准号:7301731);广东省科技计划项目(2007B011000007);广东东莞科技发展项目;中国博士后基金(批准号:20070410854)资助

摘　　要：基于食品安全性的要求,在涉及食品工程菌株的基因工程改造中不能使用抗生素或抗药性标记,无标记遗传育种法是这一领域的有益尝试,但由于缺乏一种快捷的筛选方法,转化子的筛选成为该方法的瓶颈。通过对传统的羧肽酶Y活性验证和实验室独创的96孔板结合菌落PCR两种筛选方法进行了无抗生素标记的酿酒酵母转化子筛选并对这2种方法进行比较。结果表明,在酵母菌株尚处于杂合基因的状态下,羧肽酶Y活性验证无效;通过96孔板结合菌落PCR的筛选方法可以实现高通量筛选。According to food safety requirements, antibiotic marker could not be used in gene-engineering of cells concerning food, and marker-free genetic breeding method is a meaningful try in this field. However, lack of a high-throughput selection method （the selection oftransformants） became an obstacle for this method. In this paper, two activity assay of carboxypeptidase Y and the selecting method of 96-well plate combined with colony PCR were used and compared in the selection of transformants of Saccharomyces cerevisiae with no marker. The results showed that under the condition of being a heterozygote ofS. cerevisiae, transformants selection via assay of carboxypeptidase Y was invalid and high-throughput selection could be carded out via the method of 96-well plate combined with colony PCR which was developed in the lab.

关 键 词：微生物 酿酒酵母 基因敲除 转化子 高通量筛选 PCR 

分 类 号：TS261.11[轻工技术与工程—发酵工程]