Abstract
The integration and structure of a transgene locus can have profound effects on the level and stability of transgene expression. We screened 28 transgenic birch (Betula platyphylla Suk.) lines transformed with an insect-resistance gene (bgt) using Agrobacterium tumefaciens. Among the transgenic plants, the copy number of transgene varied from one to four. A rearrangement or partial deletion had occurred in the process of T-DNA integration. T-DNA repeat formation, detected by reverse primer PCR, was found among randomly screened transgenic lines. Sequencing of the junctions between the T-DNA inserts revealed deletions of 19–589 bp and an additional 45 bp filler DNA sequence was inserted between the T-DNA repeats at one junction. Micro-homologous sequences (1–6 bp) were observed in the junctions between the T-DNA inserts. Using SiteFinding-PCR, a relatively high percentage of AT value was found for the flanking regions. Deletion of the right border repeat was observed in 12/18 of the T-DNA/plant junctions analyzed. The number of nucleotides deleted varied from 3 to 712. Deletions of 17–89 bp were observed in all left T-DNA/plant junctions analyzed. A vector backbone DNA sequence in the transgene loci was also detected using primer pairs outside the left and right T-DNA borders. Approximately 89.3% of the lines contained some vector backbone DNA. These observations revealed that it is important to check the specificity of the integration. A mechanism of T-DNA transport and integration is proposed for this long-lived tree species.
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Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (NO: 30471413 and 30872045) and Specialized Research Fund for the Doctoral Program of Higher Education (NO: 200802251038) and China Postdoctoral Science Foundation (NO: 20090460071).
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Communicated by J. Carlson.
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Zeng, FS., Zhan, YG., Zhao, HC. et al. Molecular characterization of T-DNA integration sites in transgenic birch. Trees 24, 753–762 (2010). https://doi.org/10.1007/s00468-010-0445-6
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DOI: https://doi.org/10.1007/s00468-010-0445-6