EMS对穿梭质粒pZ189诱变作用的初步研究

Mutagenesis Study of Ethylmethane Sulfonate with Shuttle Vector Plasmid pZ189

用甲基磺酸乙酯(EMS)处理穿梭质粒pZ189的转化菌,提取经处理的质粒再转化大肠杆菌MBM7070菌株。然后,在特定的选择性培养基上筛选出pZ189质粒上靶基因SupF发生了突变的转化子,计算了突变型频率。用DNA温度梯度凝胶电泳(TGGE)检出了含有SupF突变基因的DNA片段.

Shuttle vector plasmid pZ189was used as a molecular tool and the SupFinserted in the plasmid was worked as atarget gene for mutagenesis study. Thehost cells (E. coli MBM 7070) with pZ189were treated with ethylmethane sulfonate(EMS) and plated on the selective mediacontaining X-gal (5-bromo-4-chloro-3-indo-lyl-β-D-galactoside) and IPTG (isopropyl-β-D-thiogalactoside). The SupF and theLacZ amber mutant carried by the host cellscomplemented each other and thus madethe colonies blue on the selective media.However the colonies derived from theSupF mutants changed the colour fromblue to white. The mutant frequenciesin a series of experiments with differentconcentrations of EMS were estimated.Furthermore, the DNA isolated from 5SupF mutants was digested with restruic-tion enzyme Hha I. It sug gest that the214 bp Hha I fragments containing mutantSupF could be distinguished from theirwild type counterparts by temperaturegradient gel electrophoresis under optimalconditions.