Koide Tie, da Silva Neto José F, Gomes Suely L, Marques Marilis V
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 687, 05508-900 São Paulo, SP, Brazil.
Curr Microbiol. 2004 Apr;48(4):247-50. doi: 10.1007/s00284-003-4189-z.
To overcome the difficulty in obtaining mutants of the citrus strains of Xylella fastidiosa, we evaluated mutagenesis using the transposome system as a tool for the isolation of a large number of mutants. Electroporation of a commercial transposome system in X. fastidiosa CVC (Citrus Variegated Chlorosis) strain J1a12 yielded an efficiency of 1.2 x 10(3) kanamycin (Km)-resistant clones per microg of DNA. Southern blot analysis demonstrated that the transposon was randomly inserted, and nucleotide sequence analysis indicated the presence of 9 bp direct repeats flanking the transposon insertion site. Analysis by PCR of one of the insertion mutants (clone J15) showed that the transposon was stable after eight passages in solid media. These results show that the transposome system can be used to generate a random mutant library of Xylella fastidiosa CVC strain.
为克服获得木质部难养菌柑橘菌株突变体的困难，我们评估了使用转座体系统作为分离大量突变体工具的诱变方法。将一种商业转座体系统电穿孔导入木质部难养菌柑橘杂色黄化病（CVC）菌株J1a12，每微克DNA产生1.2×10³个抗卡那霉素（Km）克隆的效率。Southern印迹分析表明转座子是随机插入的，核苷酸序列分析表明转座子插入位点两侧存在9 bp的直接重复序列。对其中一个插入突变体（克隆J15）进行PCR分析表明，该转座子在固体培养基中传代八次后是稳定的。这些结果表明，转座体系统可用于构建木质部难养菌CVC菌株的随机突变文库。
