Department of Plant Molecular Biology, Molbas Research Group, State University of Leiden, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands.
Plant Mol Biol. 1985 Jul;5(4):223-34. doi: 10.1007/BF00020640.
The T-DNA structure and organization in tissues obtained via transformation of tobacco protoplasts with Ti-plasmid DNA was found to be completely different from the T-DNA introduced via Agrobacterium tumefaciens. It is often fragmented. Overlapping copies of T-DNA, having various sizes, as well as separated fragments of T-DNA were detected. The border sequences of 23 basepairs (bp), flanking the T-region in the Ti-plasmid as direct repeats are not used as preferred sequences for integration. Similar results were obtained with a T-region clone lacking one of the TL-borders. This clone, which carried the cytokinin locus and only the right border sequence of TL and the left border sequence of TR, still had the capacity to transform protoplasts. Also the Vir-region of the Ti-plasmid is not required for integration of foreign DNA via DNA transformation. This is demonstrated by the results with the T-region clone mentioned and by the transforming capacity of a Ti-plasmid carrying a mutated Vir-region. Nevertheless, in a number of Ti-plasmid DNA transformants Vir-region fragments were found to be stably integrated. Furthermore, it has been established that co-transformation can occur with plant cells. Besides the detection of Ti-plasmid fragments from outside the T-region also DNA sequences originating from two DNA sources, which were both independently present in transformation experiments, have been found in some DNA transformants, e.g. calf thymus DNA, which was used as carrier DNA. No expression of the co-transferred DNA was observed. In total three phenotypical classes of DNA transformants were isolated. Although the T-DNA was often scrambled, polyA(+) mRNA studies indicated that the different phenotypes studied can be explained by the presence of active T-DNA genes with known functions.
通过烟草原生质体转化 Ti 质粒 DNA 获得的 T-DNA 结构和组织与通过根癌农杆菌引入的 T-DNA 完全不同。它通常是碎片化的。检测到 T-DNA 的重叠副本，具有各种大小，以及分离的 T-DNA 片段。侧翼 T 区的 23 个碱基对（bp）边界序列在 Ti 质粒中作为直接重复使用，不作为整合的首选序列。在缺乏 TL 边界之一的 T 区克隆中也获得了类似的结果。该克隆携带细胞分裂素基因座，仅携带 TL 的右边界序列和 TR 的左边界序列，仍然具有转化原生质体的能力。Ti 质粒的 Vir 区也不需要通过 DNA 转化整合外源 DNA。这通过前面提到的 T 区克隆的结果以及携带突变 Vir 区的 Ti 质粒的转化能力得到证明。然而，在许多 Ti 质粒 DNA 转化体中发现 Vir 区片段稳定整合。此外，已经确定共转化可以与植物细胞发生。除了检测到 T 区以外的 Ti 质粒片段外，还在一些 DNA 转化体中发现了源自两个 DNA 来源的 DNA 序列，这两个 DNA 来源在转化实验中都是独立存在的，例如小牛胸腺 DNA，它被用作载体 DNA。没有观察到共转移 DNA 的表达。总共分离出三种表型类别的 DNA 转化体。尽管 T-DNA 经常混乱，但多 A(+)mRNA 研究表明，研究的不同表型可以通过存在具有已知功能的活性 T-DNA 基因来解释。
