甲基磺酸乙酯对穿梭质粒P^(Z189)的诱变作用及温度梯度凝胶电泳对SUP^F突变基因的检测

MUTAGENESiS STUDY OF EMS TO SHUTTLE PLASMID PZ189 AND THE MUTANT SUPF GENE DETECTION BY TGGE TECHNIQUE

穿梭质粒P^(z189)转化原核细胞后,用甲基磺酸乙酯对转化的原核细胞进行诱变处理,使P^(z189)发生突变,突变的靶基因导致了再转化克隆在选择培养基上颜色改变。用温度梯度凝胶电泳技术检测突变子上的靶基因发现,当选择的试验条件合适时,这项技术能够检测出结构变异的DNA片段。

A shuttle plasmid PZ1989 was used as a molecular tool of mutagenesis and the SupF gene, which is inserted in the plasmid, as a target in our experiments. The primary results from prokaryotic cell was reported .The Escherichia coli strain MBL7070 transformed by PZ189 was treated with mutagen Ethylmethane sulforate(EMS) and plated on the media containing X-gal (5-brome-4-chloro-3- indolyl-β-D-galactoside) and IPTG (isopropyl-β-D-thiogalac-topyranoside). The expression of SupF can complement the lacz tRNA amber mutation carried by the host cell MBM7070 and enable the bacterial to decompose lactose.That makes the colonies blue on the media containing X-gal and IPTG .However, once mutations take place in the target gene SupF of PZ189, the colonies derived from mutants will change the colour from blue to white .By this way, the mutant frequences in a series of experiment group with designed concentration of EMS can be studied. Furthermore, the treated shuttle plasmid DNA from five mutant colonies were isolated and digested with restruction enzyme Hha I .It is suggested that the 124bp HhaI fragment which is included in the mutant SupF gene can be detected from the wild counterpart by the means of temperature gradient gel electrophoresis (TGGE) under the optimal conditions.