Construction of Disarmed Ti Plasmids Transferable between Escherichia coli and Agrobacterium Species
Construction of Disarmed Ti Plasmids Transferable between Escherichia coli and Agrobacterium Species
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DOI：
10.1128/aem.01856-08
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发表时间：
2009-04-01
影响因子：
4.4
通讯作者：
Suzuki, Katsunori
中科院分区：
文献类型：
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作者：
Kiyokawa, Kazuya;Yamamoto, Shinji;Suzuki, Katsunori
农杆菌介导的植物转化已被广泛应用，但也有一些植物对这种类型的转化表现出顽固的态度。这种转化方法使用携带修饰的肿瘤诱导(Ti)质粒(缺乏转移DNA(T-DNA)区(解除武装的Ti质粒))的细菌菌株。开发能够扩大寄主范围的菌株是可取的。大量的农杆菌菌株还没有进行测试，以确定它们是否可以用于转化。为了改进解除武装的方法，获得解除解除武装的菌株，为植物转化试验做好准备，我们开发了一种简单的方案，使某些钛质粒解除武装，同时在大肠杆菌中保持，并在大肠杆菌和农杆菌之间进行动员。为了在诺帕林型钛质粒中建立该方案，将左边界序列左侧的相邻片段、PTI-Sakura右边界序列右侧的相邻片段、含有这两个片段之间的pSC101复制基因的盒、广泛宿主范围的IncP型ORIT和庆大霉素抗性基因插入自杀型SACB载体中。当使用工具质粒时，用pTiC58和PTI-Sakura中的盒替换T-DNA的频率很高，准确率很高。我们证实，修饰后的Ti质粒在大肠杆菌S17-1 lambda pir中保持稳定，并从大肠杆菌向无钛农杆菌菌株进行接合转移，重组农杆菌菌株能够将DNA转移到植物细胞中。由于改进的质粒传递系统简单高效，菌株转化为解除武装类型的菌株很容易，应该适用于筛选有用菌株的研究。
Agrobacterium-mediated plant transformation has been used widely, but there are plants that are recalcitrant to this type of transformation. This transformation method uses bacterial strains harboring a modified tumor-inducing (Ti) plasmid that lacks the transfer DNA (T-DNA) region (disarmed Ti plasmid). It is desirable to develop strains that can broaden the host range. A large number of Agrobacterium strains have not been tested yet to determine whether they can be used in transformation. In order to improve the disarming method and to obtain strains disarmed and ready for the plant transformation test, we developed a simple scheme to make certain Ti plasmids disarmed and simultaneously maintainable in Escherichia coli and mobilizable between E. coli and Agrobacterium. To establish the scheme in nopaline-type Ti plasmids, a neighboring segment to the left of the left border sequence, a neighboring segment to the right of the right border sequence of pTi-SAKURA, a cassette harboring the pSC101 replication gene between these two segments, the broad-host-range IncP-type oriT, and the gentamicin resistance gene were inserted into a suicide-type sacB-containing vector. Replacement of T-DNA with the cassette in pTiC58 and pTi-SAKURA occurred at a high frequency and with high accuracy when the tool plasmid was used. We confirmed that there was stable maintenance of the modified Ti plasmids in E. coli strain S17-1 lambda pir and conjugal transfer from E. coli to Ti-less Agrobacterium strains and that the reconstituted Agrobacterium strains were competent to transfer DNA into plant cells. As the modified plasmid delivery system was simple and efficient, conversion of strains to the disarmed type was easy and should be applicable in studies to screen for useful strains.