A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression
Song Chen A B C , Christopher A. Helliwell A , Li-Min Wu A , Elizabeth S. Dennis A , Narayana M. Upadhyaya A , Ren Zhang B , Peter M. Waterhouse A and Ming-Bo Wang A DA CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia.
B School of Biological Sciences, University of Wollongong, NSW 2522, Australia.
C Current address: Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
D Corresponding author. Email: Ming-Bo.Wang@csiro.au
Functional Plant Biology 32(8) 671-681 https://doi.org/10.1071/FP05072
Submitted: 23 March 2005 Accepted: 5 May 2005 Published: 3 August 2005
Abstract
Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d / r) or inverted-repeat (i / r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i / r T-DNA insertions is a primary inducer of transgene silencing in plants.
Keywords: Agrobacterium tumefaciens, hairpin RNA, inverted repeat, plant transformation, T-DNA vector, transgene silencing.
Acknowledgments
We thank Qianhao Zhu for the RB-nest5 primer, Louisa Matthew for critical reading of the manuscript, and Jim Peacock and Bill Taylor for their support of the work.
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