Kanamycin rescue: A simple technique for the recovery of T-DNA flanking sequences
Kanamycin rescue: A simple technique for the recovery of T-DNA flanking sequences
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DOI：
10.1007/bf02684900
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发表时间：
1996-06-01
影响因子：
2.1
通讯作者：
Camilleri, C
中科院分区：
文献类型：
--
作者：
Bouchez, D;Vittorioso, P;Camilleri, C
我们设计了一种从拟南芥T-DNA标记系中恢复T-DNA侧翼序列的新方法。由于目前使用的大多数转化载体都含有卡那霉素抗性的植物选择标记，我们可以利用T-DNA中nptII编码区的3‘端部分来补充Tn5中的细菌nptII基因的5’端，从而在大肠杆菌中构建一个功能性的卡那霉素抗性基因。我们已经构建了一个包含nptII基因5‘端的载体，该基因从Tn5一直延伸到唯一的Pst I位点。通过克隆转化株系的总DNA，我们能够直接选择含有重组功能卡那霉素基因的T-DNA片段和插入附近的拟南芥基因组DNA片段的克隆。该系统回收了长达4kb的侧翼序列。
We have designed a new method for the recovery of T-DNA flanking sequences from T-DNA-tagged Lines of Arabidopsis thaliana. Since most transformation vectors in use contain a plant-selectable marker for kanamycin resistance, we can use the 3' part of the nptII coding region from the T-DNA to complement the bacterial 5' region of the nptII gene from Tn5 to reconstruct a functional kanamycin-resistance gene in Escherichia coli. We have constructed a vector that contains the 5' part of the nptII gene from Tn5 up to the unique Pst I site. By cloning total DNA from transformed lines in this vector, we were able to select directly for clones containing a T-DNA fragment, which reconstitutes a functional kanamycin gene, and a fragment of arabidopsis genomic DNA adjacent to the insertion. Flanking sequences up to 4 kb were rescued by this system.