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  • Title: Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events.
    Author: Herman L, Jacobs A, Van Montagu M, Depicker A.
    Journal: Mol Gen Genet; 1990 Nov; 224(2):248-56. PubMed ID: 2177527.
    Abstract:
    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.
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