Changes to the core and flanking sequences of G‐box elements lead to increases and decreases in gene expression in both native and synthetic soybean promoters

: Cis-regulatory elements in promoters are major determinants of binding specificity of transcription factors (TFs) for transcriptional regulation. To improve our understanding of how these short DNA sequences regulate gene expression, synthetic promoters consisting of both classical (CACGTG) and variant G-box core sequences along with different flanking sequences derived from the promoters of three different highly expressing soybean genes, were constructed and used to regulate a green fluorescent protein (gfp) gene. Use of the classical 6-bp G-box provided information on the base level of GFP expression while modifications to the 2-4 flanking bases on either side of the G-box influenced the intensity of gene expression in both transiently transformed lima bean cotyledons and stably transformed soybean hairy roots. The proximal 2-bp sequences on either flank of the G-box significantly affected G-box activity, while the distal 2-bp flanking nucleotides also influenced gene expression albeit with a decreasing effect. Manipulation of the upstream 2- to 4-bp flanking sequence of a G-box variant (GACGTG), found in the proximal region of a relatively weak soybean glycinin promoter, significantly enhanced promoter activity using both transient and stable expression assays, if the G-box variant was first converted into a classical G-box (CACGTG). In addition to increasing our understanding of regulatory element composition and structure, this study shows that minimal targeted changes in native promoter sequences can lead to enhanced gene expression, and suggests that genome editing of the promoter region can result in useful and predictable changes in native gene expression.