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

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.