Wirtz U, Schell J, Czernilofsky A P
DNA. 1987 Jun;6(3):245-53. doi: 10.1089/dna.1987.6.245.
A chimeric neomycin phosphotransferase II (NPT II) gene, which normally provides kanamycin resistance to transformed plant cells, was inactivated by in vitro deletions. Repair plasmids not containing plant-specific transcription signals but containing only the NPT II coding region (or parts of it) were used in co-transformation experiments involving direct DNA uptake into protoplasts isolated from Nicotiana tabacum. Recombination, or gene conversion mediated by homologous sequences produced active NPT II genes in about 1% of transformants, rendering these cells resistant to kanamycin. Analysis of the size of the active enzyme indicated that recombination had occurred producing an NPT II gene indistinguishable from the wild-type gene. Southern blot analysis revealed that the bulk of co-transformed donor plasmid DNA had suffered structural modifications; however, kanamycin resistance was inherited in a Mendelian fashion, indicating that at least one functional and structurally intact copy of the regenerated NPT II gene is integrated into the host genome.
一种嵌合的新霉素磷酸转移酶II（NPT II）基因，通常赋予转化植物细胞卡那霉素抗性，通过体外缺失使其失活。不含植物特异性转录信号但仅包含NPT II编码区（或其部分）的修复质粒用于共转化实验，该实验涉及将DNA直接导入从烟草分离的原生质体中。同源序列介导的重组或基因转换在约1%的转化体中产生了活性NPT II基因，使这些细胞对卡那霉素具有抗性。对活性酶大小的分析表明发生了重组，产生了一个与野生型基因无法区分的NPT II基因。Southern印迹分析显示，大部分共转化的供体质粒DNA发生了结构修饰；然而，卡那霉素抗性以孟德尔方式遗传，这表明再生的NPT II基因至少有一个功能和结构完整的拷贝整合到了宿主基因组中。
