de Vries J, Wackernagel W
Genetik, Fachbereich Biologie, Universität Oldenburg, Germany.
Mol Gen Genet. 1998 Apr;257(6):606-13. doi: 10.1007/s004380050688.
We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6 x 10(6)-fold excess of plant DNA. Only 18 ng of potato DNA (2.5 x 10(3) genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification (e.g. by PCR).
我们开发了一种新型系统，用于灵敏检测nptII基因（卡那霉素抗性决定因子），包括转基因植物基因组中存在的此类基因。该检测方法基于一个nptII基因的重组修复，该基因在细菌启动子下游的质粒上有一个内部10碱基对的缺失。通过转化摄取nptII基因可恢复卡那霉素抗性。在大肠杆菌中，通过电穿孔提供的无启动子nptII基因在RecA依赖的重组过程中能高效挽救。为了挽救植物染色体DNA中存在的nptII基因，该系统适用于自然转化，自然转化有利于线性DNA的摄取。当用携带nptII作为标记基因的各种转基因植物（马铃薯、烟草、甜菜、油菜、番茄）的DNA转化含有质粒上突变nptII基因的感受态不动杆菌属BD413（以前称为醋酸钙不动杆菌）细胞时，获得的卡那霉素抗性转化体大致与植物DNA中nptII基因的浓度成比例。nptII基因的挽救在植物DNA过量超过6×10⁶倍的情况下发生。仅需18 ng马铃薯DNA（2.5×10³个基因组当量，每个含有一个nptII拷贝）就能产生一个卡那霉素抗性转化体。这些实验以及其他使用从土壤样品中分离的DNA的实验表明，该系统无需事先进行DNA扩增（例如通过PCR），就能可靠且高度灵敏地监测转基因植物DNA以及环境来源（如土壤）DNA中的nptII基因。
