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

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.

Induccion de Anilina Hidroxilasa por la Hormona Adrenocorticotropica y la Dexametasona / Induction of Aniline Hydroxilase by the Adenocorticotropic Hormone and Dexametasone

La administracion in vivo de la hormona adrenocorticotropica (ACTH) produce induccion de las oxidasas de funcion mixta (OFM) microsomales en el higado de raton si se utiliza anilina como sustrato. El comportamiento inductivo semeja el patron natural, pues muestra una induccion temprana con disminucion posterior como respuesta a una sola dosis de hormona, a diferencia de lo observado comunmente para otro tipo de inductores. El suministro progresivo y creciente de la hormona presenta un efecto de saturacion. La administracion del glucocorticoide dexametasona reproduce los resultados obtenidos con ACTH, lo cual esta de acuerdo con la hipotesis de que el efecto es mediado por la corteza suprarrenal. La inhibicion del efecto inductivo por cicloheximida sugiere la sintesis de novo de proteina enzimatica