Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants
Electron. j. biotechnol
;
15(4): 2-2, July 2012. ilus, tab
Article
in English
| LILACS
| ID: lil-646952
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 (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.
Full text:
Available
Index:
LILACS (Americas)
Main subject:
Oxidoreductases
/
Polymerase Chain Reaction
/
Brassica napus
/
Glycine
Language:
English
Journal:
Electron. j. biotechnol
Journal subject:
Biotechnology
Year:
2012
Type:
Article
Affiliation country:
Iran
Institution/Affiliation country:
Baqiyatallah Medical Science University/IR
/
National Institute of Genetic Engineering and Biotechnology/IR
Similar
MEDLINE
...
LILACS
LIS