High-frequency T-DNA-mediated gene tagging in plants.

Abstract

An insertion element [transferred DNA (T-DNA)], transferred by soil agrobacteria into the nuclear genome of plants, was used for induction of gene fusions in Arabidopsis thaliana, Nicotiana tabacum, and Nicotiana plumbaginifolia. A promoterless aph(3'')II (aminoglycoside phosphotransferase II) reporter gene was linked to the right end of the T-DNA and tranformed into plants along with a plasmid replicon and a selectable hygromycin-resistance gene. Transcriptional and translational reporter gene fusions were identified by screening for APH(3'')II enzyme activity in diverse tissues of transgenic plants. The frequency of gene fusions, estimated by determination of the copy number of T-DNA insertions, showed that on average 30% of T-DNA inserts induced gene fusions in Arabidopsis and Nicotiana. Gene fusions were rescued from plants by transformation of the T-DNA-linked plasmid and flanking plant DNA into Escherichia coli. By dissection of gene fusion and construction of chimeric genes, callus- and root-specific promoters were identified that showed an altered tissue specificity in the presence of a 3''-downstream-located 35S promoter. Transcript mapping of a gene fusion and expression of a non-frame transcriptional fusion of bacterial luciferase luxA and luxB genes demonstrated that dicistronic transcripts are translated in tobacco.