CUP1为筛选标记的酿酒酵母整合型多基因表达载体的构建  

Construction of an Integrated Multi-gene Expression Vector of Saccharomyces cerevisiae Using CUP1 as Selection Marker

作　　者：阳辛凤[1,2,3] 徐林[2] 弓淑芳[2] 郭安平[2] 孔华[2] 贺立卡[2] 

机构地区：[1]海南大学农学院,海口570228 [2]中国热带农业科学院热带生物技术研究所,海口571101 [3]中国热带农业科学院分析测试中心,海口571101

出　　处：《生物技术通报》2011年第10期139-144,共6页Biotechnology Bulletin

基　　金：国家"863"重点课题(2007AA021307);转基因生物新品种培育重大专项课题(2008ZX08012-04)

摘　　要：为了赋予工业酿酒酵母对淀粉和纤维素的降解活性,提高酿酒酵母对粗木薯粉进行酒精发酵时的酒精产率;另一方面,为了解决工业酿酒酵母不适于使用营养缺陷型筛选标记对转化子进行筛选的问题,以及避免引入抗药性标记基因带来的安全性问题,构建了以抗铜蛋白基因CUP1为筛选标记的酿酒酵母整合型多基因表达载体。以载体pYES2-PMF-rDNA为基础,以新的筛选标记基因CUP1替换原有的尿嘧啶Ura-基因,得到载体pYES2M。再顺序插入葡聚糖内切酶基因eg3、葡萄糖淀粉酶基因ga1和β-葡萄糖苷酶基因bgl1,构建得到以CUP1为筛选标记的酵母整合型三价表达载体pYES2M-eg3-ga1-bgl1,其中每个基因都具有独立而完整的表达盒,包括启动子、信号肽和终止子,从而实现多基因单表达载体一次转化。In order to make industrial Saccharomyces cerevisiae possessing degradation function to starch and cellulose,improve alcohol yield as alcohol fermentation on crude cassava by Saccharomyces cerevisiae,resolve the problem of auxotrophic selection marker not fitting for the screening of industrial S.cerevisiae transformants and biosafety of drug-resistant marker genes,a yeast integrated multi-gene expression vector was constructed using the copper resistance gene CUP1 as selection marker.Based on the vector pYES2-PMF-rDNA,the original Ura-gene was replaced by the new selection marker CUP1 and the new vector was named as pYES2M.Glucanase gene eg3,glucoamylase gene ga1 and β-glucosidase gene bgl1 was inserted into pYES2M and a new yeast trivalent integrated expression vector pYES2M-eg3-ga1-bgl1 was constructed,in which CUP1 was selection marker.In the vector pYES2M-eg3-ga1-bgl1,each gene has an independent and complete expression cassette,including promoter,signal peptide and the terminator.The study proved that transformation of multi-gene by one vector could be realized.

关 键 词：酿酒酵母 载体 CUP1 多基因转化 

分 类 号：TS261.11[轻工技术与工程—发酵工程]