PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the Arabidopsis genome.
BACKGROUND
The large serine recombinase phiC31 from broad host range Streptomyces temperate phage, catalyzes the site-specific recombination of two recognition sites that differ in sequence, typically known as attachment sites attB and attP. Previously, we characterized the phiC31 catalytic activity and modes of action in the fission yeast Schizosaccharomyces pombe.


RESULTS
In this work, the phiC31 recombinase gene was placed under the control of the Arabidopsis OXS3 promoter and introduced into Arabidopsis harboring a chromosomally integrated attB and attP-flanked target sequence. The phiC31 recombinase excised the attB and attP-flanked DNA, and the excision event was detected in subsequent generations in the absence of the phiC31 gene, indicating germinal transmission was possible. We further verified that the genomic excision was conservative and that introduction of a functional recombinase can be achieved through secondary transformation as well as manual crossing.


CONCLUSIONS
The phiC31 system performs site-specific recombination in germinal tissue, a prerequisite for generating stable lines with unwanted DNA removed. The precise site-specific deletion by phiC31 in planta demonstrates that the recombinase can be used to remove selectable markers or other introduced transgenes that are no longer desired and therefore can be a useful tool for genome engineering in plants.
Thomson
James G
JG
Crop Improvement and Utilization Research Unit, Western Regional Research Center, USDA-ARS, 800 Buchanan Street, Albany, CA 94710, USA. James.Thomson@ars.usda.gov
Chan
Ronald
R
Thilmony
Roger
R
Yau
Yuan-Yeu
YY
Ow
David W
DW
D017360:Arabidopsis / Q000235:genetics*; D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology*; D018744:DNA, Plant / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D018745:Genome, Plant; D016297:Mutagenesis, Site-Directed; D030821:Plants, Genetically Modified / Q000235:genetics; D011401:Promoter Regions, Genetic; D045522:Recombinases / Q000235:genetics*; D011995:Recombination, Genetic
D018744:DNA, Plant; D045522:Recombinases