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

Chen, Song, Helliwell, Christopher A., Wu, Li-Min, Dennis, Elizabeth S., Upadhyaya, Narayana M., Zhang, Ren, Waterhouse, Peter M., & Wang, Ming-Bo (2005) A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression. Functional Plant Biology, 32(8), pp. 671-681.

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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. © CSIRO 2005.

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6 citations in Web of Science®

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ID Code: 65955
Item Type: Journal Article
Refereed: Yes
Keywords: Agrobacterium tumefaciens, Hairpin RNA, Inverted repeat, Plant transformation, T-DNA vector, Transgene silencing, Bacteriology, Cell culture, Genes, RNA, DNA, plant breeding, Cells, Nucleic Acids, Tobacco, Agrobacterium, Arabidopsis, Nicotiana tabacum, Rhizobium
DOI: 10.1071/FP05072
ISSN: 1445-4408
Subjects: Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > GENETICS (060400)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > PLANT BIOLOGY (060700)
Divisions: Current > Schools > School of Earth, Environmental & Biological Sciences
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Copyright 2005 C S I R O Publishing
Deposited On: 09 Jan 2014 04:19
Last Modified: 30 Jan 2014 00:46

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