Characterization of the rolB promoter on mikimopine-type pRi1724 T-DNA

The promoter activity of the rolB gene encoded on mikimopine-type pRi1724 (1724rolB) was elucidated. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that 1724rolB mRNA was highly accumulated in roots of 1724rolB-transformed tobacco plants. A β-glucuronidase (GUS) fluorometric assay using a 1724rolB promoter-GUS construct also showed high promoter activity in the roots. A GUS histochemical assay revealed that 1724rolB was expressed in the vicinity of root apical meristematic cells of the vascular cylinder in the main roots, in lateral root primordial and meristematic cells, in veins of cotyledons, and faintly in the shoot apex. Since the product of 1724rolB has a N-terminal stretch 17 amino acid longer than the rolB of agropine-type pRi1855 (1855rolB), primer extension analysis was used to show that the transcription initiation point of 1724rolB was located beyond the putative TATA box of 1855rolB, suggesting that the locations of the minimal promoters of the two rolB genes differed from each other. 1724rolB promoter analysis using deletion constructs indicated that the minimal region necessary for this promoter activity was, at most, −215 bp upstream of the translation initiation point. A predominant auxin-responsive sequence was located at position −313 to −216 upstream of the transcription initiation point, containing the Nicotiana tabacum rolB domain B factor 1 (NtBBF1) binding motif ACTTTA.