Brumbley Stevens M, Petrasovits Lars A, Birch Robert G, Taylor Paul W J
Bureau of Sugar Experiment Stations, Indooroopilly, Queensland, Australia.
Mol Plant Microbe Interact. 2002 Mar;15(3):262-8. doi: 10.1094/MPMI.2002.15.3.262.
Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp. xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally. Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/microg of plasmid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/microg using cosmid cloning vectors pLAFR3 and pLAFR5-km. The transformation/transposition frequency was 0 to 70 CFU/microg of DNA, using suicide vectors pUCD623 and pSUP2021 containing transposable elements Tn4431 and Tn5, respectively. It was necessary to grow Lxx in media containing 0.1% glycine for electroporation and to amplify large plasmids in a dam-/dcm- E. coli strain and purify the DNA by anion exchange. To keep selection pressure at an optimum, the transformants were grown on nitrocellulose filters (0.2-microm pore size) on media containing the appropriate antibiotics. Transposon Tn4431 containing a promoterless lux operon from Vibrio fischeri and a tetracycline-resistance gene was introduced on the suicide vector pUCD623. All but 1% of the putative transposon mutants produce light, indicating transposition into functional Lxx genes. Southern blot analysis of these transformants indicates predominantly single transposon insertions at unique sites. The cosmid cloning vector pLAFR5-km was stably maintained in Lxx. The development of a transformation and transposon mutagenesis system opens the way for molecular analysis of pathogenicity determinants in Lxx.
已开发出用于木糖雷夫松氏菌木糖亚种（Lxx）遗传转化和插入诱变的条件，Lxx是宿根矮化病（RSD）的致病病原体，RSD是国际上甘蔗最具破坏性和最难处理的病害之一。使用棒状杆菌/大肠杆菌穿梭载体pCG188、pDM302和pDM306时，转化频率为每微克质粒DNA 1至10个菌落形成单位（CFU），使用粘粒克隆载体pLAFR3和pLAFR5-km时，转化频率为每微克50至500 CFU。使用分别含有转座元件Tn4431和Tn5的自杀载体pUCD623和pSUP2021时，转化/转座频率为每微克DNA 0至70 CFU。有必要在含有0.1%甘氨酸的培养基中培养Lxx用于电穿孔，并在dam-/dcm-大肠杆菌菌株中扩增大型质粒，通过阴离子交换纯化DNA。为了将选择压力保持在最佳状态，将转化体在含有适当抗生素的培养基上的硝酸纤维素滤膜（孔径为0.2微米）上培养。含有来自费氏弧菌的无启动子lux操纵子和四环素抗性基因的转座子Tn4431被导入自杀载体pUCD623。除1%外，所有推定的转座子突变体均产生光，表明转座到功能性Lxx基因中。对这些转化体的Southern印迹分析表明，主要是在独特位点的单转座子插入。粘粒克隆载体pLAFR5-km在Lxx中稳定维持。转化和转座子诱变系统的开发为Lxx中致病性决定因素的分子分析开辟了道路。
