The intron and 5' distal region of the soybean Gmubi promoter contribute to very high levels of gene expression in transiently and stably transformed tissues.

KEY MESSAGE: An extended version of an intron-containing soybean polyubiquitin promoter gave very high levels of gene expression using three different validation tools. The intron-containing Glycine max polyubiquitin promoter (Gmubi) is able to regulate expression levels five times higher than the widely used CaMV35S promoter. In this study, eleven Gmubi derivatives were designed and evaluated to determine which regions contributed to the high levels of gene expression, observed with this promoter. Derivative constructs regulating GFP were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. With both expression systems, removal of the intron in the 5'UTR led to reduced levels of gene expression suggesting a role of the intron in promoter activity. Promoter constructs containing an internal intron duplication and upstream translocations of the intron resulted in higher and similar expression levels to Gmubi, respectively, indicating the presence of enhancers within the intron. Evaluation of 5' distal extensions of the Gmubi promoter resulted in significantly higher levels of GFP expression, suggesting the presence of upstream regulatory elements. A twofold increase in promoter strength was obtained when Gmubi was extended 1.5 kb upstream to generate GmubiXL (2.4 kb total length). In stably transformed soybean plants containing GFP regulated by CaMV35S, Gmubi and GmubiXL, the GmubiXL promoter clearly produced the highest levels of gene expression, with especially high GFP fluorescence in the vascular tissue and root tips. Use of GmubiXL leads to very high levels of gene expression in soybean and represents a native soybean promoter, which may be useful for regulating transgene expression for both basic and applied research.