Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants

• Faranak Hadi ^[1] ; Ali Hatef Salmanian ^[1] ; Elham Ghazizadeh ^[1] ; Jafar Amani ^[2] ; Kambiz Akbari Noghabi ^[1] ; Amir Mousavi ^[1]
  1. [1] National Institute of Genetic Engineering and Biotechnology Department of Plant Biotechnology
  2. [2] Baqiyatallah Medical Science University Applied Microbiology Research Center
• Localización: Electronic Journal of Biotechnology, ISSN-e 0717-3458, Vol. 15, Nº. 4, 2012, págs. 2-2
• Idioma: inglés
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• Resumen

  • Background: 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.