Functionality of the beta/six site-specific recombination system in tobacco and Arabidopsis: a novel tool for genetic engineering of plant genomes.
The beta recombinase is a member of the prokaryotic site-specific serine recombinases (invertase/resolvase family), which in the presence of a DNA bending cofactor can catalyse DNA deletions between two directly oriented 90-bp six recombination sites. We have examined here whether the beta recombinase can be expressed in plants and whether it displays in planta its specific catalytic activity excising DNA sequences that are flanked by six sites. In plant protoplasts, the enzyme could be expressed as a GFP-beta recombinase fusion which can localise to the cell nucleus. Beta recombinase stably expressed in tobacco plants can catalyse deletion of a spacer region that is flanked by directly oriented six sites and has been placed between promoter and a GUS reporter gene (preventing GUS expression). In transient transformation experiments, beta recombinase-mediated elimination of the spacer results in transcriptional induction of the GUS gene. Similarly, beta recombinase in stably double-transformed Arabidopsis plants deletes specifically the spacer region of a reporter construct that has been incorporated into the genome. In the segregating T1 generation, plants were identified that contain exclusively the recombined reporter construct. In summary, our results demonstrate that functional / recombinase can be expressed in plants and that the enzyme is suitable to precisely eliminate undesired sequences from plant genomes. Therefore, the beta/six recombination system (and presumably related recombinases) may become an attractive tool for plant genetic engineering.
Grønlund
Jesper T
JT
Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Stemmer
Christian
C
Lichota
Jacek
J
Merkle
Thomas
T
Grasser
Klaus D
KD
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D018745:Genome, Plant; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011523:Protoplasts / Q000201:enzymology; D045522:Recombinases / Q000737:chemistry / Q000235:genetics*; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics*
D017931:DNA Primers; D018744:DNA, Plant; D045522:Recombinases