Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
EMBO J. 1985 Feb;4(2):277-84. doi: 10.1002/j.1460-2075.1985.tb03626.x.
We have constructed a set of small vectors based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens which allow the transfer of exogenous DNA into plant chromosomes. These vectors contain: (i) a chimeric gene containing the transcriptional control signals from the nopaline synthase gene and the coding sequence for neomycin phosphotransferase; (ii) the ColE1 replicon; (iii) the cos site of bacteriophage lambda; (iv) the border sequences from the ends of the T-DNA region of the Ti plasmid; and (v) a wide host range replicon. Due to the small size of these cosmid vectors, DNA fragments up to 35 kbp can be inserted by an in vitro packaging method in Escherichia coli. The ability of these vectors to be stably replicated in both E. coli and A. tumefaciens allows their subsequent transfer to and maintenance in Agrobacterium without intermediate genetic manipulations. We demonstrate that DNA cloned into these vectors in A. tumefaciens can efficiently transform plants when in trans with a wild-type Ti plasmid which donates the functions necessary for DNA transfer and integration. We also show that only the right border of the T-DNA is necessary for DNA transformation.
我们构建了一组基于根癌农杆菌（Agrobacterium tumefaciens）肿瘤诱导（Ti）质粒的小载体，这些载体允许外源 DNA 转移到植物染色体中。这些载体包含：（i）一个嵌合基因，包含胭脂碱合成酶基因的转录控制信号和新霉素磷酸转移酶的编码序列；（ii）ColE1 复制子；（iii）噬菌体 lambda 的 cos 位点；（iv）Ti 质粒 T-DNA 区域末端的边界序列；和（v）一个广泛宿主范围的复制子。由于这些 cosmid 载体体积小，通过体外包装方法可以插入长达 35 kbp 的 DNA 片段。这些载体在大肠杆菌和根癌农杆菌中稳定复制的能力允许它们在不需要中间遗传操作的情况下随后转移到根癌农杆菌中并在其中维持。我们证明，当与野生型 Ti 质粒（其提供 DNA 转移和整合所需的功能）反式克隆到这些载体中的 DNA 时，能够有效地转化植物。我们还表明，只有 T-DNA 的右边界对于 DNA 转化是必需的。
