Characterization of T-DNA insertions in transgenic grapevines obtained by Agrobacterium-mediated transformation |
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Authors: | Giorgio Gambino Walter Chitarra Fatemeh Maghuly Margit Laimer Paolo Boccacci Daniela Torello Marinoni Ivana Gribaudo |
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Institution: | 1. Plant Virology Institute (CNR), Grugliasco Unit, Via L. da Vinci 44, 10095, Grugliasco, TO, Italy 3. Department of Arboriculture and Pomology, University of Torino, Via L. da Vinci 44, 10095, Grugliasco, TO, Italy 2. Plant Biotechnology Unit, Institute of Applied Microbiology BOKU, Nussdorfer L?nde 11, 1190, Wien, Austria
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Abstract: | T-DNA integration patterns in 49 transgenic grapevines produced via Agrobacterium-mediated transformation were analyzed. Inverse PCR (iPCR) was performed to identify T-DNA/plant junctions. Sequence comparison
revealed several deletions in the T-DNA right border (RB) and left border (LB), and filler DNA and duplications or deletions
of grapevine DNA at the T-DNA insertion loci. In 20 T-DNA/grapevine genome junctions microsimilarities were found associated
with the joining points and in all grapevine lines microsimilarities were present near the breaking points along the 30 bases
of T-DNA adjacent to the two borders. Analysis of target site preferences of T-DNA insertions indicated a non-random distribution
of the T-DNA, with a bias toward the intron regions of the grapevine genes. Compositional analysis of grapevine DNA around
the T-DNA insertion sites revealed an inverse relationship between the CG and AT-skews and AT rich sequences present at 300–500 bp
upstream the insertion points, near the RB of the T-DNA. PCR assays showed that vector backbone sequences were integrated
in 28.6% of the transgenic plants analyzed and multiple T-DNAs frequently integrated at the same position in the plant genome,
resulting in the formation of tandem and inverted repeats. |
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