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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

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Abstract

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An extended version of an intron-containing soybean polyubiquitin promoter gave very high levels of gene expression using three different validation tools.

Abstract

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.

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Acknowledgments

We wish to acknowledge Robert A. Bouchard for his helpful suggestions and construction development during the early course of this research. We would also like to thank Cheri Nemes for the generation of transgenic plants, Fei-Yi Tsai for the analysis of GFP expression in transgenic plants, Nuananong Semsang for her assistance with the generation of Gmubi derivatives in pCAMBIA1300, and Jenny Tran for the GFP quantification of GmubiM in hairy roots. We appreciate the helpful suggestions from Drs. Eric Stockinger and Leah McHale for their critical reading of this manuscript. Salaries and research support were provided by the United Soybean Board, Bayer CropScience, and by State and Federal funds appropriated to The Ohio State University/Ohio Agricultural Research and Development Center. This research was also supported, in part, through The Consortium for Plant Biotechnology Research, Inc. by DOE Prime Agreement No. DEFG36-02G012026. This support does not constitute an endorsement by DOE or by The Consortium for Plant Biotechnology Research, Inc. of the views expressed in this publication. Mention of trademark or proprietary products does not constitute a guarantee or warranty of the product by OSU/OARDC and also does not imply approval to the exclusion of other products that may also be suitable.

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Carola M. De La Torre

    Present address: Division of Plant Sciences, 315 Christopher S. Bond Life Sciences Center, University of Missouri-Columbia, Columbia, MO, 65211, USA

Authors and Affiliations

  1. Department of Horticulture and Crop Science, OARDC/The Ohio State University, 1680 Madison Ave, Wooster, OH, 44691, USA

    Carola M. De La Torre & John J. Finer

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  1. Carola M. De La Torre
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  2. John J. Finer
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Correspondence to John J. Finer.

Additional information

Communicated by Kathryn K. Kamo.

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De La Torre, C.M., Finer, J.J. 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. Plant Cell Rep 34, 111–120 (2015). https://doi.org/10.1007/s00299-014-1691-7

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  • DOI: https://doi.org/10.1007/s00299-014-1691-7

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