Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events

Mol Gen Genet. 1990 Nov;224(2):248-56. doi: 10.1007/BF00271558.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Blotting, Southern
  • Chromosome Mapping
  • Conjugation, Genetic
  • DNA / genetics
  • DNA / isolation & purification
  • DNA, Bacterial / genetics*
  • Genes, Bacterial
  • Genetic Markers
  • Kanamycin Kinase
  • Molecular Sequence Data
  • Phosphotransferases / genetics
  • Plants / genetics*
  • Plasmids
  • Promoter Regions, Genetic
  • Restriction Mapping
  • Rhizobium / genetics*
  • Transformation, Genetic

Substances

  • DNA, Bacterial
  • Genetic Markers
  • T-DNA
  • DNA
  • Phosphotransferases
  • Kanamycin Kinase