Abstract
The expression profile of a natural bi-directional promoter, derived from the Brassica napus EPSPS-A gene, was studied in transgenic soybean (Glycine max C.V. Maverick) lines. Two constructs, pDAB100331 and pDAB100333, were assembled to test the bi-directionality of the promoter. Two reporter genes, gfp and gusA, were employed and they were interchangeably placed in both constructs, one on each end of the promoter such that both proteins expressed divergently in each construct. In the T0 generation, GUS expression was more uniform throughout the leaf of pDAB100333 transgenic plants, where the gusA gene was expressed from the downstream or EPSPS-A end of the bi-directional promoter. Comparatively, GUS expression was more localized in the midrib and veins of the leaf of pDAB100331 transgenic plants, where the gusA gene was expressed from the upstream end of the bi-directional promoter. These observations indicated a unique expression pattern from each end of the promoter and consistently higher expression in genes expressed from the downstream end (e.g., EPSPS-A end) of the promoter in the tissues examined. The GFP expression pattern followed that of GUS when placed in the same position relative to the promoter. In the T1 generation, transcript analysis also showed higher expression of both gusA and gfp when those genes were located at the downstream end of the promoter. Accordingly, the pDAB100331 events exhibited a higher gfp/gusA transcript ratio, while pDAB100333 events produced a higher gusA/gfp transcript ratio consistent with the observations in T0 plants. These results demonstrated that the EPSPS-A gene bidirectional promoter can be effectively utilized to drive expression of two transgenes for the desired traits.
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Abbreviations
- GUS (protein, or gusA gene):
-
β-Glucuronidase
- GFP:
-
Green fluorescent protein
- YFP:
-
Yellow fluorescent protein
- EPSPS-A:
-
5-enolpyruvyl shikimate-3-phosphate synthase gene A
- BEAP:
-
Brassica napus EPSPS-A promoter
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Acknowledgements
The authors acknowledge the excellent technical assistance of Sara Bennett and Sarah Worden for cloning and/or vector construction. The authors are grateful to Lyudmila Sidorenko for the use of Brassica napus expression atlas, to Rodrigo Sarria, Cory Christensen, Michelle Smith, Dayakar Pareddy and Nagesh Sardesai for critical review of the manuscript, and to Pon Samuel for identification of cell types in GUS stained tissues. We are also thankful to Rodrigo Sarria for his support during this project.
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Chennareddy, S., Cicak, T., Clark, L. et al. Expression of a novel bi-directional Brassica napus promoter in soybean. Transgenic Res 26, 727–738 (2017). https://doi.org/10.1007/s11248-017-0042-1
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DOI: https://doi.org/10.1007/s11248-017-0042-1