Rosana L, Suhartono M T, Suwanto A
Laboratory for Biochemistry and Microbiology IUC Biotechnology IPB, Bogor Agricultural University, Indonesia.
Mol Biotechnol. 1999 Apr;11(2):129-35. doi: 10.1007/BF02915806.
Protease negative mutant of Xanthomonas campestris pathovar glycine 8ra (prt-mutant) was constructed by mutagenesis employing artificial transposon Omegon-Km. Transposon delivery was conducted through diparental conjugation using X. campestris pathovar glycine 8ra as recipient and Escherichia coli S17-1 carrying pJFF 3500 plasmid as the donor. The frequency of transconjugation was found 1.9 x 10(-7) per recipient. Enzyme analysis indicated the presence of mutant with lower protease activity than that of the wild-type. Genetic analysis employing pulsed-field gel electrophoresis (PFGE) of the genomic DNA digested with AseI or SpeI restriction endonuclease could significantly differentiate X. campestris pathovar glycine 8ra prt from the wild-type parent. The 9.85 kb pLR omega 6 plasmid was constructed from the genomic DNA of the prt mutant after being digested with KpnI restriction endonuclease and ligated with T4 DNA ligase.
利用人工转座子Omegon-Km诱变构建了野油菜黄单胞菌致病变种大豆8ra的蛋白酶阴性突变体（prt突变体）。转座子传递通过双亲接合进行，以野油菜黄单胞菌致病变种大豆8ra作为受体，携带pJFF 3500质粒的大肠杆菌S17-1作为供体。发现转接合频率为每个受体1.9×10⁻⁷。酶分析表明存在蛋白酶活性低于野生型的突变体。使用AseI或SpeI限制性内切酶消化基因组DNA后进行脉冲场凝胶电泳（PFGE）的遗传分析可以显著区分野油菜黄单胞菌致病变种大豆8ra prt与野生型亲本。9.85 kb的pLR omega 6质粒是由prt突变体的基因组DNA用KpnI限制性内切酶消化后与T4 DNA连接酶连接构建而成。
