Characterization and event specific-detection by quantitative real-time PCR of T25 maize insert.
T25 is one of the 4 maize transformation events from which commercial lines have so far been authorized in Europe. It was created by polyethylene glycol-mediated transformation using a construct bearing one copy of the synthetic pat gene associated with both promoter and terminator of the 35S ribosomal gene from cauliflower mosaic virus. In this article, we report the sequencing of the whole T25 insert and the characterization of its integration site by using a genome walking strategy. Our results confirmed that one intact copy of the initial construct had been integrated in the plant genome. They also revealed, at the 5' junction of the insert, the presence of a second truncated 35S promoter, probably resulting from rearrangements which may have occurred before or during integration of the plasmid DNA. The analysis of the junction fragments showed that the integration site of the insert presented high homologies with the Huck retrotransposon family. By using one primer annealing in the maize genome and the other in the 5' end of the integrated DNA, we developed a reliable event-specific detection system for T25 maize. To provide means to comply with the European regulation, a real-time PCR test was designed for specific quantitation of T25 event by using Taqman chemistry.
Collonnier
Cécile
C
Laboratoire de Méthodologies de la Détection des OGM, Institut National de la Recherche Agronomique, Route de Saint Cyr, Versailles, France.
Schattner
Alexandra
A
Berthier
Georges
G
Boyer
Francine
F
Coué-Philippe
Géraldine
G
Diolez
Annick
A
Duplan
Marie-Noëlle
MN
Fernandez
Sophie
S
Kebdani
Naïma
N
Kobilinsky
André
A
Romaniuk
Marcel
M
de Beuckeleer
Marc
M
de Loose
Marc
M
Windels
Pieter
P
Bertheau
Yves
Y
D024509:3' Flanking Region / Q000235:genetics; D024506:5' Flanking Region / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D005062:European Union; D030841:Food, Genetically Modified; D007881:Legislation, Food; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012639:Seeds / Q000737:chemistry; D003313:Zea mays / Q000737:chemistry* / Q000235:genetics*
D017931:DNA Primers; D018744:DNA, Plant