Kanamycin rescue: A simple technique for the recovery of T-DNA flanking sequences

被引:29
作者
Bouchez, D [1 ]
Vittorioso, P [1 ]
Courtial, B [1 ]
Camilleri, C [1 ]
机构
[1] INRA,BIOL CELLULAIRE LAB,F-78026 VERSAILLES,FRANCE
关键词
arabidopsis; insertional mutagenesis; T-DNA; kanamycin;
D O I
10.1007/BF02684900
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:115 / 123
页数:9
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