摘要
以穿梭质粒pCN60为载体,大肠杆菌C600为受体构建了扣囊拟内孢霉(Endomycopsis fibuligera)G45 Sau 3A基因文库。从基因文库中提取重组质粒DNA并转化酿酒酵母(Saccharomyces cerevisiae)BJ1991,选出四个具有o-淀粉酶活性的转化子,琼脂糖凝胶电泳结果证实插入的DNA片段为9.0kb。对插入DNA片段亚克隆,确定。-淀粉酶基因位于PstI-Sall 3.9kb片段上,启动子位于PstI-EcoRI 的1.3kb片段上。用亚克隆PGK11.9kb片段置换。-淀粉酶启动子区,其转化子的e-淀粉酶活性有明显提高。
Endomycopsis fibuligera G45 genomic library was established in E. colt C600 with E. coli-yeast shuttle plasmid vector pCN60. Four transformants with amylolytic enzyme activity were screened by transforming recipient strain S. cerevisiae BJ1991 with the hybrid plasmid from the genimic library. Gel electro-phoresis results showed that the size of the inserted DNA fragments was 7,0kb. Subcloning of the inserted DNA fragments showed that the cloning gene was located in the Pstl-Sall 3.9kb fragments, the promoter region was in the Pstl-EcoRI 1.3 kb fragments. By replacement of the promoter of the amylolytic enzyme with a strong promoter PGK1 (1.9kb) fragment, the amylolytic enzyme activity was higher than that of the original transformants (pCN60(G45)).
出处
《微生物学报》
CAS
CSCD
北大核心
1995年第6期404-409,共6页
Acta Microbiologica Sinica
基金
国家自然科学基金资助项目
