| Journal: | Electronic journal of biotechnology |
| Database: | PERIÓDICA |
| System number: | 000359296 |
| ISSN: | 0717-3458 |
| Authors: | Hadi, Faranak1 Salmanian, Ali Hatef1 Ghazizadeh, Elham1 Amani, Jafar2 Noghabi, Kambiz Akbari1 Mousavi, Amir1 |
| Institutions: | 1National Institute of Genetic Engineering and Biotechnology, Tehran, Iran, Department of Plant Biotechnology, Teherán. Irán 2Baqiyatallah Medical Science University, Applied Microbiology Research Center, Teherán. Irán |
| Year: | 2012 |
| Volumen: | 15 |
| Number: | 4 |
| Country: | Chile |
| Language: | Inglés |
| Document type: | Artículo |
| Approach: | Experimental, aplicado |
| English abstract | For successful in vitro plant regeneration, plant cell lines with multiple transgene integration and low transgene expression levels need to be ruled out. Although real-time polymerase chain reaction (RT-PCR) is a rapid way to accomplish this, it is also expensive and typically limits the size of the target sequence. Quantitative competitive PCR (QC-PCR) is proven to be a safe and accurate method for determination of both copy number and quantification of transcript levels of synthetic transgenes in transformed plants. Results: The glyphosate oxidoreductase genewas chemically synthesized and used to transform Brassica napus L. via Agrobactrium-mediated transformation. A construct containing the mutated form of a synthetic glyphosate oxidoreductase (gox) gene (internal standard) was prepared. Gene copy number was estimated in nine independent transgenic lines using QC-PCR as well as the standard method of Southern blot analysis. By quantitative RT-PCR, transcript levels were also determined in these lines. High (> 3), medium to high (2.2-3), medium to low (1-2.2), and low (< 1) levels of transcript were detected. Conclusions: No direct relationship was found between copy number and transgene expression levels. QC-PCR method could be implemented to screen putative transgenic plants and quickly select single T-DNA inserts. QC-PCR methods and the prepared competitor construct may be useful for future quantification of commercial transgenic food and feed |
| Disciplines: | Biología, Química |
| Keyword: | Angiospermas, Genética, Biotecnología, Reacción en cadena de la polimerasa (PCR), Plantas transgénicas, Transformación genética, Transcripción génica, Brassica napus |
| Keyword: | Biology, Chemistry, Angiosperms, Genetics, Biotechnology, Polymerase chain reaction (PCR), Transgenic plants, Genetic transformation, Gene transcription, Brassica napus |
| Full text: | Texto completo (Ver HTML) |
