Bailey M J, Lilley A K, Thompson I P, Rainey P B, Ellis R J
Natural Environment Research Council, Institute of Virology and Environmental Microbiology, Oxford, UK.
Mol Ecol. 1995 Dec;4(6):755-63. doi: 10.1111/j.1365-294x.1995.tb00276.x.
A plasmid-free, non-pathogenic, ribosomal RNA group 1 fluorescent pseudomonad, Pseudomonas fluorescens SBW25, was selected from the microflora of sugar beet (Beta vulgaris) and modified to contain constitutively expressed marker genes. By site directed homologous recombination a KX cassette [kanamycin resistance (kanr) and catechol 2,3 dioxygenase (xylE)] and a ZY cassette [lactose utilization (lacZY, beta-galactosidase, lactose permease)] were introduced at least 1 Mbp apart on the 6.6 Mbp bacterial chromosome. Separate sites were selected to provide sensitive detection methods and allow assessments of marker gene stability of the genetically modified micro-organism (GMM), SBW25EeZY6KX, when it colonized the leaves and roots of sugar beet plants following seed inoculation.
从甜菜（Beta vulgaris）的微生物区系中筛选出一种无质粒、非致病性的核糖体RNA 1组荧光假单胞菌——荧光假单胞菌SBW25，并对其进行改造，使其含有组成型表达的标记基因。通过定点同源重组，在6.6 Mbp的细菌染色体上至少相隔1 Mbp引入了一个KX盒[卡那霉素抗性（kanr）和儿茶酚2,3-双加氧酶（xylE）]和一个ZY盒[乳糖利用（lacZY，β-半乳糖苷酶，乳糖通透酶）]。选择不同的位点以提供灵敏的检测方法，并在转基因微生物SBW25EeZY6KX通过种子接种在甜菜植株的叶和根上定殖时，评估其标记基因的稳定性。
