枯草芽孢杆菌bgIS基因在酵母染色体上整合

Bacillus Subtilis bglS Gene Integrated into Chromsomes of Saccharomyces cerevisiae

将枯草杆菌β-1,3-1,4-葡聚糖酶基因(bglS)2.7kbEcoRI片段和酵母染色体rDNA片段克隆到整合型不含酵母自主复制序列(Autonomusly replicating sequence)的大肠杆菌/酵母菌穿梭质粒YIP5上,构建成YIP5-bglS-rDNA的杂种质粒PCZH,转化S.cerevisiae并得到表达。稳定性测定表明,Y33(PCZH101)和Y33(PCZH104)在无选择压力的YEPD培养基中繁殖70代以上,两个转化子90％以上的酵母细胞仍含有质粒并且遗传特征与染色体行为相似。以bglS基因作探针,与酵母染色体DNA的Southern bloot分子杂交证实bglS基因已整合到酵母菌染色体上。

YIP5 without automomusly replicating sequence of yeast was employed for an E. coli/S. cerevisiae shuttle vector. A rDNA fragment of yeast and a 2. 7kb EcoRI DNA fragment earring a Bacillus subtilis endo-β-1, 3-1, 4-glucanase gene (bglS) from E. coli plasmid YCSH1 was cloned into the vector to construct a hybrid plasmid PCZH1 with YIP5-bglS-rDNA genes. The hybrid plasmid was used to transform the S. cerevisiae and express the β-1, 3-1, 4-glueanase gene. The stability tests of Y33 (YCSH1), Y33 (PCZHlOl)and Y33(PCZH104)showed that the two integrated transformants were stabler than the episomid transformant in yeast cells. The cells of the two integrated transformants retained their plasmids more than 90 percent over a period of 70 generations of growth in batch culture under non-selective conditions. At the same time the genetic character of the hybrid plasmid seemed as the yeast chromosome. The Southern blot hy-brization between the yeast DNA and the bglS gene probe with 32P indicated that the bglS gene was integrated into the chromosomes of yeast.