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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 15, No. 4, 2012, pp. 1-14
Bioline Code: ej12037
Full paper language: English
Document type: Research Article
Document available free of charge

Electronic Journal of Biotechnology, Vol. 15, No. 4, 2012, pp. 1-14

 en Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants
Hadi, Faranak; Salmanian, Ali Hatef; Mousavi, Amir; Ghazizadeh, Elham; Amani, Jafar & Noghabi, Kambiz Akbari

Abstract


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 (real-time 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 gene was chemically synthesized and used to transform Brassica napus check for this species in other resources L. via Agrobactrium check for this species in other resources -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 competitive reverse transcriptase PCR (QC-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.

Keywords
Brassica napus L.; competitive quantitative PCR; transcript level; transgene copy number

 
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