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Open Access Highly Accessed Research article

High level transgenic expression of soybean (Glycine max) GmERF and Gmubi gene promoters isolated by a novel promoter analysis pipeline

Carlos M Hernandez-Garcia1, Robert A Bouchard1, Paul J Rushton24, Michelle L Jones1, Xianfeng Chen35, Michael P Timko2 and John J Finer1*

Author Affiliations

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

2 Department of Biology, University of Virginia, Charlottesville, VA 22904 USA

3 Department of Microbiology, University of Virginia Health Systems, Charlottesville, VA 22908 USA

4 Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007 USA

5 USACE, Environmental Lab, ERDC, 3909 Halls Ferry Road, Vicksburg, MS 39180 USA

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BMC Plant Biology 2010, 10:237  doi:10.1186/1471-2229-10-237

Published: 4 November 2010

Abstract

Background

Although numerous factors can influence gene expression, promoters are perhaps the most important component of the regulatory control process. Promoter regions are often defined as a region upstream of the transcriptional start. They contain regulatory elements that interact with regulatory proteins to modulate gene expression. Most genes possess their own unique promoter and large numbers of promoters are therefore available for study. Unfortunately, relatively few promoters have been isolated and characterized; particularly from soybean (Glycine max).

Results

In this research, a bioinformatics approach was first performed to identify members of the Gmubi (

    G.
    m
ax
    ubi
quitin) and the GmERF (
    G
.
    m
ax
    E
thylene
    R
esponse
    F
actor) gene families of soybean. Ten Gmubi and ten GmERF promoters from selected genes were cloned upstream of the gfp gene and successfully characterized using rapid validation tools developed for both transient and stable expression. Quantification of promoter strength using transient expression in lima bean (Phaseolus lunatus) cotyledonary tissue and stable expression in soybean hairy roots showed that the intensity of gfp gene expression was mostly conserved across the two expression systems. Seven of the ten Gmubi promoters yielded from 2- to 7-fold higher expression than a standard CaMV35S promoter while four of the ten GmERF promoters showed from 1.5- to 2.2-times higher GFP levels compared to the CaMV35S promoter. Quantification of GFP expression in stably-transformed hairy roots of soybean was variable among roots derived from different transformation events but consistent among secondary roots, derived from the same primary transformation events. Molecular analysis of hairy root events revealed a direct relationship between copy number and expression intensity; higher copy number events displayed higher GFP expression.

Conclusion

In this study, we present expression intensity data on 20 novel soybean promoters from two different gene families, ubiquitin and ERF. We also demonstrate the utility of lima bean cotyledons and soybean hairy roots for rapid promoter analyses and provide novel insights towards the utilization of these expression systems. The soybean promoters characterized here will be useful for production of transgenic soybean plants for both basic research and commercial plant improvement.