Nugent G D, Ten Have M, van der Gulik A, Dix P J, Uijtewaal B A, Mordhorst A P
Biology Department, National University of Ireland, Maynooth, County Kildare, Ireland.
Plant Cell Rep. 2005 Aug;24(6):341-9. doi: 10.1007/s00299-005-0930-3. Epub 2005 Jun 18.
Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEG-mediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.
利用两个载体获得了番茄质体转化体，这两个载体含有烟草或龙葵的克隆质体DNA，并包含赋予壮观霉素和链霉素抗性的点突变。通过聚乙二醇介导的原生质体直接摄取DNA，随后在含有壮观霉素的培养基上进行筛选，从而获得转化体。通过绘制限制酶位点图谱证实，16个株系含有该点突变。与自发的壮观霉素抗性突变体相比，对用每个载体获得的一个株系进行了更详细的分析。通过对反向重复区域中质体DNA靶向区域的序列分析，证实了克隆的茄属或烟草质体DNA通过多次重组事件整合到番茄质体基因组中。幼苗中壮观霉素和链霉素抗性或敏感性的母系遗传也证实了这两个转化体的转质体系状态。结果证明了一种选择策略在番茄中的有效性，该策略避免了显性细菌抗生素抗性基因的整合。
