Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants
Figure 6
Gene targeting of the Arabidopsis PPO locus. (a) Schematic representation of gene targeting at the PPO locus. The grey box marked PPO represents the PPO coding region, and the thick black lines represent flanking plant genomic DNA. The repair T-DNA [10] (LB for left border and RB for right border) contains a BAR (black box marked BAR) selection marker linked to the 5′ truncated ΔPPO gene (grey box marked ΔPPO). The thin lines indicate the T-DNA sequences. The two mutations conferring butafenacil resistance are indicated by asterisks. The probes and the restriction enzyme digestion sites used for Southern blot analysis and the sizes of expected bands are shown. (b) Southern blotting of progeny plants of three butafenacil resistant lines (III, IV and V). DNA was digested with NcoI and KpnI and separated in 0.7% agarose gel along a positive gene-targeted sample (lane P), a wild-type sample (lane W) and dig-labeled Lambda HindIII marker (lane M), blotted and hybridized with a DIG-labelled 5′PPO probe (upper panels), 3′PPO probe (middle panels) and a BAR probe (lower panels). The bands with sizes expected after gene targeting are indicated with 3′PPO and 5′PPO. The wild-type PPO band (WT) and bands representing additional T-DNAs are also shown.
