Novel Constructions to Enable the Integration of Genes into the Agrobacterium tumefaciens C58 Chromosome
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 pBluescri...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
The American Phytopathological Society
2001-04-01
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| Series: | Molecular Plant-Microbe Interactions |
| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/MPMI.2001.14.4.577 |
| Summary: | 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 incPα 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 β-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 β-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. |
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| ISSN: | 0894-0282 1943-7706 |