Characterization of cis-acting sequences regulating root-specific gene expression in tobacco.

The expression of the tobacco root-specific gene TobRB7 was characterized. Gel blot hybridizations to RNA isolated from various tobacco tissues demonstrated that steady-state TobRB7 mRNA is not detected in expanded leaf, stem, or shoot apex tissue. To determine the spatial pattern of expression, in situ hybridization to root sections revealed that TobRB7 expression is localized to root meristem and immature central cylinder regions. The 5' flanking region of the gene was studied with respect to its ability to direct root-specific expression. Deletions of 5' flanking sequence were fused to the beta-glucuronidase (GUS) reporter gene and transformed into tobacco. Our data demonstrated that sequences 636 base pairs from the site of transcription initiation are sufficient to direct the root-specific GUS expression in transgenic tobacco, whereas sequences 299 base pairs from the site of transcription initiation fail to direct root-specific expression. A negative regulatory element was apparent between 813 base pairs and 636 base pairs 5' of the transcription initiation site. Histochemical localization of GUS activity in transgenic plants was consistent with in situ hybridization results: GUS activity was localized to the root meristem and central cylinder regions. GUS activity appeared 2 days post-germination in the primary root meristem. In lateral roots, GUS activity was detected from the time of initiation.