Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events.
During Agrobacterium tumefaciens infection, the T-DNA flanked by 24 bp imperfect direct repeats is transferred and stably integrated into the plant chromosome at random positions. Here we measured the frequency with which a promoterless reporter gene is activated after insertion into the Nicotiana tabacum SR1 genome. When adjacent to the right or left T-DNA border sequences, at least 35% of the transformants express the marker gene, suggesting preferential T-DNA insertion (greater than 70%) in transcriptionally active regions of the plant genome. When the promoterless neomycin phosphotransferase II (nptII) gene is located internally in the T-DNA, the activation frequency drops to 1% since gene activation requires T-DNA truncation. These truncation events in the nptII upstream region occur independently of the nature of the upstream sequence and of the T-DNA length. Deletion of the right border region prevents the detection of activated marker genes. Therefore, T-DNA truncation probably occurs after synthesis of a normal T-DNA intermediate during the transfer and/or integration process. In the absence of border regions, expression of the nptII selectable marker directed by the nopaline synthase promoter was detected in 1 out of 10(5) regenerated calli, suggesting the possibility that any DNA sequence from the Ti plasmid can be transformed into the plant genome, albeit at a low frequency.
Herman
L
L
Laboratorium voor Genetica, Rijksuniversiteit Gent, Belgium.
Jacobs
A
A
Van Montagu
M
M
Depicker
A
A
D001483:Base Sequence; D015139:Blotting, Southern; D002874:Chromosome Mapping; D003227:Conjugation, Genetic; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial; D005819:Genetic Markers; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000235:genetics; D010944:Plants / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012231:Rhizobium / Q000235:genetics*; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D005819:Genetic Markers; C036483:T-DNA; D004247:DNA; D010770:Phosphotransferases; D019868:Kanamycin Kinase