Figure 3

Rescue of regulatory and coding regions directly into a plant transformation vector and development of a Gateway Expression vector. To rescue the regulatory and coding regions of a gene, the plant transformation vector is amplified by PCR using primers that incorporate 50 bp of homology corresponding to sequences 5' and 3' of the promoter and terminator regions. The vector amplicon is transformed into an E. coli strain possessing a BAC containing the genomic region of interest and expressing the bacteriophage lambda Red homologous recombination proteins. Selection for the plant transformation vector identifies clones containing the rescued gene which can be directly used to transform plants using A. tumefaciens. To create a novel Gateway Expression vector incorporating the 5' and 3' regulatory regions of the rescued gene, the Gateway Destination cassette is first amplified by PCR using primers that incorporate 50 bp of homology corresponding to sequences 3' of the promoter and 5' of the terminator regions. The Destination cassette amplicon is transformed into an E. coli strain possessing the cloned gene in a plant transformation vector and expressing the bacteriophage lambda Red homologous recombination proteins. Selection for the plant transformation vector and the marker on the Destination cassette identifies clones where the original cloned coding region is replaced by the Destination cassette. The Destination cassette now flanked by the cognate 5' and 3' regulatory regions of the initial cloned gene can be used to link reporter genes or other genes of interest to the cloned regulatory regions.