Department of Biology, Washington University, 63130, St. Louis, MO, USA.
Plant Cell Rep. 1993 Jan;12(3):133-8. doi: 10.1007/BF00239093.
Tobacco plants were transformed with derivatives of a binary vector pMON505 and two kanamycin resistant lines that were nopaline positive were selected for second transformation. The plasmids used for the second transformation were derivatives of pMON850 which carries the nopaline synthase gene in addition to a gene for gentamicin resistance. Insertion of each transgene was confirmed by Southern hybridization. Surprisingly, we found that more than 50% of the doubly transformed tobacco plants were nopaline negative. Tobacco plants that were transformed only by the second vector exhibited nopaline accumulation. DNA methylation patterns at the HpaII site in the promoter region of the nopaline synthase gene did not correlate with the nopaline phenotype. In some plant lines, seedlings of the R1 generation which segregated out the second T-DNA insertion recovered the nop(+) phenotype. These results indicate that nopaline accumulation was inhibited by the presence of the second T-DNA.
烟草植株用二元载体 pMON505 的衍生物转化，选择出对卡那霉素有抗性且能产生胭脂碱的两个转化株系进行第二轮转化。第二轮转化使用的质粒是 pMON850 的衍生物，该质粒除了携带新霉素抗性基因外，还携带胭脂碱合酶基因。通过 Southern 杂交验证了每个转基因的插入。令人惊讶的是，我们发现超过 50%的双转化烟草植株胭脂碱含量为阴性。只经第二轮载体转化的烟草植株表现出胭脂碱积累。在胭脂碱合酶基因启动子区 HpaII 位点的 DNA 甲基化模式与胭脂碱表型没有相关性。在一些植物株系中，分离出第二轮 T-DNA 插入的 R1 代幼苗恢复了 nop(+)表型。这些结果表明，第二个 T-DNA 的存在抑制了胭脂碱的积累。
