A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

Functional Plant Biology : FPB
Song ChenMing-Bo Wang

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 r...Continue Reading

References

Sep 1, 1995·The Plant Journal : for Cell and Molecular Biology·Y G LiuR F Whittier
Mar 9, 1999·The Plant Journal : for Cell and Molecular Biology·S J Clough, A F Bent
Apr 29, 1999·Cell·E U Selker
Oct 26, 1999·Trends in Plant Science·P M WaterhouseM B Wang
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Jun 27, 2003·Cellular and Molecular Life Sciences : CMLS·A S Pickford, C Cogoni
Mar 9, 2005·Current Opinion in Plant Biology·Ming-Bo Wang, Michael Metzlaff

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Citations

Mar 1, 2006·TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik·Narayana M UpadhyayaElizabeth S Dennis
Jun 14, 2019·Epigenetics : Official Journal of the DNA Methylation Society·Ulrike SchumannMing-Bo Wang

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Datasets Mentioned

BETA
AB015474
AB016882

Methods Mentioned

BETA
transgenic
phosphotransferase
PCR
TAIL-PCR

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