Design of a novel system for the construction of vectors for Agrobacterium-mediated plant transformation.
The loxP-Cre site-specific recombination system of phage P1 was used to develop a novel strategy to construct cointegrate vectors for Agrobacterium-mediated plant transformation. A pTi disarmed helper plasmid (pAL1166) was constructed by replacing the oncogenic T-DNA by a loxP sequence and a spectinomycin resistance marker in the octopine-type pTiB6 plasmid. The cre gene was cloned into an unstable incP plasmid. A third plasmid, which did not replicate in Agrobacterium and contained another loxP sequence together with a kanamycin resistance marker, was used to test the system. Electroporation of this third plasmid into an Agrobacterium strain harbouring both pAL1166 and the Cre-encoding plasmid resulted in kanamycin-resistant cells containing a cointegrate between pAL1166 and the incoming plasmid. Cointegration occurred by Cre-mediated recombination at the loxP sites, and the cointegrate was stabilized in the Agrobacterium cells by the loss of the Cre-encoding plasmid shortly after the recombination event had taken place.
Mozo
T
T
Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University, The Netherlands.
Hooykaas
P J
PJ
D017101:Bacteriophage P1 / Q000235:genetics; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004277:DNA, Single-Stranded; D005822:Genetic Vectors; D019426:Integrases; D008969:Molecular Sequence Data; D010944:Plants / Q000235:genetics*; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics; D011995:Recombination, Genetic; D012231:Rhizobium / Q000235:genetics*; D014170:Transformation, Genetic; D014764:Viral Proteins
D004277:DNA, Single-Stranded; D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D011938:Rec A Recombinases