Identification of cis-Acting Elements Important for Expression of the Starch-Branching Enzyme I Gene in Maize Endosperm

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Abstract
The genes encoding the starch-branching enzymes (SBE) SBEI, SBEIIa, and SBEIIb in maize (Zea mays) are differentially regulated in tissue specificity and during kernel development. To gain insight into the regulatory mechanisms controlling their expression, we analyzed the 5′-flanking sequences of Sbe1 using a transient gene expression system. Although the 2.2-kb 5′-flanking sequence between −2,190 and +27 relative to the transcription initiation site was sufficient to promote transcription, the addition of the transcribed region between +28 and +228 containing the first exon and intron resulted in high-level expression in suspension-cultured maize endosperm cells. A series of 5′ deletion and linker-substitution mutants identified two critical positivecis elements, −314 to −295 and −284 to −255. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from maize kernels interact with the 60-bp fragment containing these two elements. Expression of the Sbe1 gene is regulated by sugar concentration in suspension-cultured maize endosperm cells, and the region −314 to −145 is essential for this effect. Interestingly, the expression of mEmBP-1, a bZIP transcription activator, in suspension-cultured maize endosperm cells resulted in a 5-fold decrease in Sbe1 promoter activity, suggesting a possible regulatory role of the G-box present in theSbe1 promoter from −227 to −220.