new challenges in the production and analysis of recombinant DNA. Large numbers of
promoters and genes encoded by these genomes have been discovered, but many
remain uncharacterized, providing an incentive to design and construct vectors with the
capacity for high-throughput functional analysis. Traditional ligase-mediated cloning is
Figure 7.17.Plant gene expression vectors for conventional cloning using restriction digestion and
ligation (a), GatewayTMrecombination cloning (b), and univector recombination cloning (c). The
first vectors shown in (a), (b), and (c) are designed to allow a gene to be ectopically expressed in a
plant cell. The second vectors shown for each category contain the GFP (green fluorescent protein)
gene. These vectors are designed to effect protein fusions with GFP to help identify the subcellular
target of a protein under investigation. Ideally, three vectors for each type are frequently made, one
for each reading frame, to ensure that a perfect fusion between the GOI and the marker gene is
made. The insert DNA must be in an “open” ORF configuration (described in the text) so that no
stop codon is present between the GOI and the marker gene.
178 RECOMBINANT DNA, VECTOR DESIGN, AND CONSTRUCTION