Isolation of tobacco DNA segments with plant promoter activity

We constructed a promoter probe vector, pGVL120, to isolate plant DNA segments with promoter activity in tobacco. Plant nuclear DNA Sau3A fragments were inserted in front of the npt-II sequence, and a mixture of recombinant plasmids was mobilized to Agrobacterium sp. and used to transform tobacco protoplasts. By kanamycin selection, transformed plant cell lines containing NPT-II T-DNAs were isolated. Eight of these cell lines were regenerated and analyzed for the levels of NPT-II activity in stem, root, midrib, and leaf. These levels demonstrated novel regulation patterns in each isolate. One cell line, T20, was analyzed in detail and found to contain four different T-DNAs. One of the recloned T-DNAs, T20-2, contains an insert of 401 base pairs in front of the NPT-II sequence, and by reintroducing this T-DNA into plant cells we could demonstrate that this insert provides a promoter sequence. The NPT-II enzyme activity under the control of the P20 promoter is especially high in stem and root, but low in leaf and callus, both in the originally isolated T20 plant and in independently isolated transformants with the T20-2 T-DNA.