Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers
Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers
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DOI：
10.1046/j.1365-313x.1996.10010165.x
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发表时间：
1996-07-01
期刊：
影响因子：
7.2
通讯作者：
Kumashiro, T
中科院分区：
文献类型：
--
作者：
Komari, T;Hiei, Y;Kumashiro, T
构建了携带两个独立 T-DNA 的新型“超级二元”载体。一种 T-DNA 含有耐药性选择标记基因，另一种含有 β-葡萄糖醛酸酶 (GUS) 基因。携带该载体的根癌农杆菌 LBA4404 产生了大量的烟草 (Nicotiana tabacum L.) 和水稻 (Oryza sativa L.) 转化子。两种T-DNA 共转化的频率大于47%。从超过一半的共转化子中获得了 GUS 阳性、药物敏感的后代，通过 Southern 杂交和聚合酶链反应进行的分子分析证实了 T-DNA 的整合和分离。因此，与选择标记在遗传上可分离的不可选择的T-DNA被整合到超过四分之一的初始耐药转化体中。由于可以通过简单的程序将各种DNA片段插入不可选择的T-DNA中，因此这些载体可能对于生产不同植物物种的无标记转化体非常有用。还测试了将两种 T-DNA 从根癌农杆菌混合物递送至植物，但共转化的频率相对较低。
Novel 'super-binary' vectors that carried two separate T-DNAs were constructed. One T-DNA contained a drug-resistance, selection-marker gene and the other contained a gene for beta-glucuronidase (GUS). A large number of tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.) transformants were produced by Agrobacterium tumefaciens LBA4404 that carried the vectors. Frequency of co-transformation with the two T-DNAs was greater than 47%. GUS-positive, drug-sensitive progeny were obtained from more than half of the co-transformants, Molecular analyses by Southern hybridization and polymerase chain reactions confirmed integration and segregation of the T-DNAs. Thus, the non-selectable T-DNA that was genetically separable from the selection marker was integrated into more than a quarter of the initial, drug-resistant transformants. Since various DNA fragments may be inserted into the non-selectable T-DNA by a simple procedure, these vectors will likely be very useful for the production of marker-free transformants of diverse plant species. Delivery of two T-DNAs to plants from mixtures of A. tumefaciens was also tested, but frequency of cotransformation was relatively low.