A bifunctional selectable marker gene for T-DNA tagging of plant promoters
Plant promoters are the principle cis-acting regulatory sequences responsible for the temporal and spatial expression of genes. One method for isolating plant promoters is based on the ability of a common soil bacterium, Agrobacterium tumefaciens , to transfer a specific segment of DNA (T-DNA) into plant cells. This specific T-DNA has been shown to integrate stably into the recipient plant genome. If the T-DNA contains a promoterless marker gene, then T-DNA integration events occurring adjacent and downstream to a promoter region can be detected by the activation of the marker gene. These T-DNA-mediated gene fusions, consisting of an unknown plant promoter sequence and the coding sequence of a marker gene, can be isolated using the marker gene as a promoter tag. The key objective of this work was to develop a novel, bifunctional selectable marker gene and assess its use as: a selectable marker gene in bacterial and plant transformation systems, and as a promoter tag for T-DNA promoter-tagging studies in dicots. A bifunctional fusion gene was produced between phosphinothricin acetyltransferase and neomycin phosphotransferase (PAT::NPT II), by fusing an NPT II coding sequence to the 3' terminus of the PAT gene. The PAT gene product confers tolerance to a non-selective herbicide L-phosphinothricin (Ignite™, Hoechst AG). The neomycin phosphotransferase ('npt II') gene allows for direct selection of transformed cells with the antibiotic, kanamycin. Using an in vivo Escherichia coli selection system, a translational fusion gene between these two reporter genes was achieved. The resulting protein had activities of both parent enzymes. This was demonstrated both in transformed Escherichia coli and in transformed Nicotiana tabacum and Brassica napus plants. Using this bifunctional selectable marker gene, a T-DNA promoter tagging vector, pBAU2, was constructed and its utility was demonstrated in Nicotiana tabacum. One of the N. tabacum promoter tagged events was selected for subsequent promoter isolation studies. The promoter from this regenerant was isolated by screening a Lambda subgenomic library and also by thermal asymmetric interlaced (TAIL-)PCR. The isolated upstream regulatory sequence was fused to a reporter gene, â-glucuronidase ('gus'), and subjected to a preliminary evaluation in Nicotiana tabacum and in Brassica napus.
pat::nptII, plant promoters, marker genes, fusion genes, transgenic plants, crop science, phosphinothricin acetyltransferase, neomycin phosphotransferase II
Doctor of Philosophy (Ph.D.)