Identification of plant promoters in situ by T-DNA-mediated transcriptional fusions to the npt-II gene.

We have constructed an Agrobacterium-mediated plant vector,pHTT88,which upon integration into the plant chromosomes can produce transcriptional fusions between upstream control elements in the plant genome and the gene for neomycin phosphotransferase II (npt-II). The vector is based on a juxtaposition of a T-DNA right border to the coding sequence of npt-II. Using this system it is possible to isolate kanamycin-resistant plant calli where the site of integration is suitably positioned downstream of plant transcription signals. We have demonstrated that the NPT-II activity of such tobacco transformants shows different tissue-specific patterns of regulation in the regenerated plants. Among eight transformants analyzed, we have isolated one gene fusion expressed only in roots and others expressed only in stem. The results demonstrate that a gene fusion vector can be used effectively in plants to identify and characterize regulatory elements.