Hansen G, Chilton M D
CIBA-Geigy Corporation, Research Triangle Park, NC 27709.
Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14978-83. doi: 10.1073/pnas.93.25.14978.
We describe a novel plant transformation technique, termed "agrolistic," that combines the advantages of the Agrobacterium transformation system with the high efficiency of biolistic DNA delivery. Agrolistic transformation allows integration of the gene of interest without undesired vector sequence. The virulence genes virD1 and virD2 from Agrobacterium tumefaciens that are required in bacteria for excision of T-strands from the tumor-inducing plasmid were placed under the control of the CaMV35S promoter and codelivered with a target plasmid containing border sequences flanking the gene of interest. Transient expression assays in tobacco and in maize cells indicated that vir gene products caused strand-specific nicking in planta at the right border sequence, similar to VirD1/VirD2-catalyzed T-strand excision observed in Agrobacterium. Agrolistically transformed tobacco calli were obtained after codelivery of virD1 and virD2 genes together with a selectable marker flanked by border sequences. Some inserts exhibited right junctions with plant DNA that corresponded precisely to the sequence expected for T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion events. We designate these as "agrolistic" inserts, as distinguished from "biolistic" inserts. Both types of inserts were found in some transformed lines. The frequency of agrolistic inserts was 20% that of biolistic inserts.
我们描述了一种新型的植物转化技术，称为“农杆菌介导基因枪转化法”，它结合了农杆菌转化系统的优点和基因枪DNA递送的高效率。农杆菌介导基因枪转化法允许整合感兴趣的基因而无不需要的载体序列。来自根癌农杆菌的毒力基因virD1和virD2，它们在细菌中是从致瘤质粒切除T链所必需的，被置于CaMV35S启动子的控制下，并与一个含有位于感兴趣基因两侧的边界序列的目标质粒共同递送。在烟草和玉米细胞中的瞬时表达分析表明，vir基因产物在植物中导致右边界序列处的链特异性切口，类似于在农杆菌中观察到的VirD1/VirD2催化的T链切除。在共同递送virD1和virD2基因以及一个两侧带有边界序列的选择标记后，获得了农杆菌介导基因枪转化的烟草愈伤组织。一些插入片段显示出与植物DNA的右连接，这与T-DNA（致瘤质粒转移到植物细胞的部分）插入事件预期的序列精确对应。我们将这些称为“农杆菌介导基因枪转化法”插入片段，以区别于“基因枪法”插入片段。在一些转化株系中发现了这两种类型的插入片段。农杆菌介导基因枪转化法插入片段的频率是基因枪法插入片段频率的20%。
