title: Novel constructions to enable the integration of genes into the Agrobacterium tumefaciens C58 chromosome.
abstract: We constructed several versatile sets of vectors that can be used to introduce any gene into the pgl/picA locus of the Agrobacterium tumefaciens C58 chromosome without affecting T-DNA transfer. One set contains a fragment containing the lacIq and lacZ genes and a multiple cloning site from pBluescriptII SK(+) inserted into a PstI site between the pgl and picA genes on an incPalpha plasmid. The resulting plasmid contains eight unique restriction endonuclease sites and the ability to use blue-white screening for the presence of an insert. A second plasmid also contains a beta-lactamase gene within this locus and provides a convenient ampicillin-carbenicillin resistance marker for the selection of genes integrated into the chromosome following double homologous recombination (homogenotization). A third plasmid contains, in addition to the lacZ, lacIq, and beta-lactamase genes within the pgl/picA locus, a sacRB gene cassette within the vector to counterselect against the presence of the vector within A. tumefaciens. To test this system, we introduced a wild-type virD2 gene into the A. tumefaciens chromosome at the pgl/picA locus. When a Ti plasmid harboring a deletion of virD2 was in this strain, the integrated virD2 gene complemented the virD2 deletion and the resulting transformation phenotype was identical to that resulting from A. tumefaciens strains harboring a wild-type virD2 gene located on a replicating plasmid.
authors: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
authors: L Y
authors: LY
authors: Lee
authors: J M
authors: JM
authors: Humara
authors: S B
authors: SB
authors: Gelvin
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D002876:Chromosomes, Bacterial; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D010947:Plants, Toxic; D010957:Plasmids; D014026:Nicotiana / Q000382:microbiology