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 th...

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Autores principales:	Hadi,Faranak, Salmanian,Ali Hatef, Ghazizadeh,Elham, Amani,Jafar, Noghabi,Kambiz Akbari, Mousavi,Amir
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2012
Materias:	Brassica napus L. competitive quantitative PCR transcript level transgene copy number
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000400002
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spelling	oai:scielo:S0717-345820120004000022012-08-22Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plantsHadi,FaranakSalmanian,Ali HatefGhazizadeh,ElhamAmani,JafarNoghabi,Kambiz AkbariMousavi,Amir Brassica napus L. competitive quantitative PCR transcript level transgene copy number 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.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.15 n.4 20122012-07-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000400002en
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Brassica napus L. competitive quantitative PCR transcript level transgene copy number
spellingShingle	Brassica napus L. competitive quantitative PCR transcript level transgene copy number Hadi,Faranak Salmanian,Ali Hatef Ghazizadeh,Elham Amani,Jafar Noghabi,Kambiz Akbari Mousavi,Amir Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants
description	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.
author	Hadi,Faranak Salmanian,Ali Hatef Ghazizadeh,Elham Amani,Jafar Noghabi,Kambiz Akbari Mousavi,Amir
author_facet	Hadi,Faranak Salmanian,Ali Hatef Ghazizadeh,Elham Amani,Jafar Noghabi,Kambiz Akbari Mousavi,Amir
author_sort	Hadi,Faranak
title	Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants
title_short	Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants
title_full	Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants
title_fullStr	Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants
title_full_unstemmed	Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants
title_sort	development of quantitative competitive pcr for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2012
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000400002
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Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants

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