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.
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.
Impact and interest:
Citation counts are sourced monthly from and citation databases.
These databases contain citations from different subsets of available publications and different time periods and thus the citation count from each is usually different. Some works are not in either database and no count is displayed. Scopus includes citations from articles published in 1996 onwards, and Web of Science® generally from 1980 onwards.
Citations counts from theindexing service can be viewed at the linked Google Scholar™ search.
|Item Type:||Journal Article|
|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|
|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|
Repository Staff Only: item control page