Transgene excision from wheat chromosomes by phage phiC31 integrase
The Streptomyces phage phiC31 integrase was tested for its ability to excise transgenic DNA from the wheat genome by site-specific recombination. Plants that stably express phiC31 integrase were crossed to plants carrying a target construct bearing the phiC31 recognition sites, attP and attB. In the progeny, phiC31 recombinase mediates recombination between the att sites of the target locus, which results in excision of the intervening DNA. Recombination events could be identified in 34 independent wheat lines by PCR and Southern blot analysis and by sequencing of the excision footprints. Recombinant loci were inherited to the subsequent generation. The results presented here establish the integrase-att system as a tool for catalysing the precise elimination of DNA sequences from wheat chromosomes.
KeywordsStreptomyces phage phiC31 integrase Site-specific recombination Transgene excision Transgenic wheat
Bacterial attachment site, phiC31 target recombination sequence
Phage attachment site, phiC31 target recombination sequence
- attR, attL
phiC31 integrase recombination products
- pICH14313, pICH13130
Vectors containing a Streptomyces phage phiC31 integrase coding sequence
- ICH14313, ICH13130
Genomic locus containing the Streptomyces phage phiC31 integrase coding sequence
Vector containing phiC31 integrase target recombination sequences
Genomic target locus carrying phiC31 integrase target recombination sequences
- ICH27371-N, ICH27371-C
Derivative genomic locus resulting from phiC31 integrase-mediated site-specific recombination
The authors are grateful to Dr. Heike Schmuths (Saaten Union Biotec Gatersleben, Germany) for logistic cooperation and Kerstin Denzin, Linda Tillack, Christin Meinhardt and Erika Grützemann for laboratory support and plant care. Furthermore, we acknowledge Wolf v. Rhade, Dr. Ralf Schachschneider (Nordsaat GmbH Böhnshausen, Germany), Dr. Jens Weyen (Saaten Union Biotec, Leopoldshöhe, Germany) and Dr. Frank Wolter (PflanzenInnovationsAgentur; PIA) for constant support. We thank anonymous reviewers for helpful comments. In particular, the authors also wish to thank Dr. Renate Schmidt for her comments on the manuscript and for many stimulating discussions. The research was done at the Leibniz Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK) Gatersleben with funding from the Bundesministerium für Bildung und Forschung (BMBF, GABI-FUTURE grant 0315043A).