Isolation of a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens

Abstract

The two most abundant transcripts derived from TR-DNA within plant cells transformed by an octopine strain of Agrobacterium tumefaciens arise from divergent transcription, both originating within an 500 bp section of the T-DNA. Using a combination of subcloning and exonuclease digestion, a 479-bp DNA fragment, directly flanked by the initiation codons for the two adjacent open reading frames, was isolated. The resulting DNA fragment was fused, in both orientations, to the neomycin phosphotransferase (NPT II) gene of the transposon Tn5 prior to introduction into Nicotiana tabacum cells via the Ti plasmid. The intergenic fragment was found to initiate expression of the NPT II gene in either orientation as assayed by kanamycin resistance of the transformed plant tissue as well as by enzymatic assay of the NPT II gene product. The plasmids described here are potential selection-expression vectors for plant systems.