A Novel Cis-Acting Element Conferring Root-Preferred Gene Expression in Maize

Screening a random oligonucleotide library using a modified differential Southwestern method, we generated a root-enriched library of candidate promoter cis-elements that interacted with maize root nuclear proteins. Three candidates that were examined formed root-specific DNA-protein complexes in electrophoretic mobility shift assays. When fused upstream to a synthetic core (SynCore) promoter driving the GUS reporter gene, only one of the three candidates, designated ROL6, generated significantly higher levels of root-preferred expression in particle bombardment-mediated transient assays compared to the SynCore promoter-driven GUS gene. The other two elements showed little or no effect on promoter activiry. The addition of the maize Ubi 1 flanking region (-865 to -54 bp) upstream of the ROL6-SynCore promoter further enhanced root-preferred expression approximately 2-fold over the high level of expression of the maize Ubi 1 promoter alone. Based on public database searches, the ROL6 sequence shows some embedded MYBbinding motifs yet very little homology to any known plant cis-acting elements and may contain one or more novel root-preferred cis-acting elements. Overall these results demonstrate first the effectiveness of using this screening approach to isolate functionally active promoter elements and second that ROL6 functions as a root-preferred activator and can be used to modify the expression pattern of transgenes.