Agrobacterium tumefaciens-Mediated Transformation of Valsa mali: An Efficient Tool for Random Insertion Mutagenesis
Figure 4
Molecular analysis of T-DNA integration into the genome of randomly chosen transformants resistant to hygromycin B. (a) Polymerase chain reaction assay with primers specific for the amplification of an internal 987 bp fragment of the hph gene. Lane P positive control with vector pBIG3C, W wild-type Valsa mali LXS240101 genomic DNA, Lanes 1–10 genomic DNA isolated from putative transformants PFL1–PFL10, and M DNA molecular weight markers with bases indicated on the left. (b) Southern blot analysis of transformants with DIG-labeled hph gene probe. P positive control with vector pBIG3C linearized with ApaI, W wild-type Valsa mali LXS240101 genomic DNA digested with HindIII, 1–6 genomic DNA isolated from putative transformants PFL1-PFL6 digested with HindIII, and M DNA molecular weight markers with bases indicated on the left. These six transformants show single copy inserts and the insertion sites are different.
