Abstract
Main Conclusion
Specific sequences within the leader intron of a soybean polyubiquitin gene stimulated gene expression when placed either within a synthetic intron or upstream of a core promoter.
The intron in the 5′ untranslated region of the soybean polyubiquitin promoter, Gmubi, seems to contribute to the high activity of this promoter. To identify the stimulatory sequences within the intron, ten different sequential intronic sequences of 40 nt were isolated, cloned as tetrameric repeats and placed upstream of a minimal cauliflower mosaic virus 35S (35S) core promoter, which was used to control expression of the green fluorescent protein. Intron fragment tetramers were also cloned within a modified, native intron, creating a Synthetic INtron Cassette (SINC), which was then placed downstream of Gmubi and 35S core promoters. Intron fragment tetramers and SINC constructs were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Intron fragments, used as tetramers upstream of the 35S core promoter, yielded up to 80 times higher expression than the core promoter in transient expression analyses and ten times higher expression in stably transformed hairy roots. Tetrameric intronic fragments, cloned downstream of the Gmubi and 35S core promoters and within the synthetic intron, also yielded increased transient and stable GFP expression that was up to 4 times higher than Gmubi alone and up to 40 times higher than the 35S core promoter alone. These intron fragments contain sequences that seem to act as promoter regulatory elements and may contribute to the increased expression observed with this native strong promoter. Intron regulatory elements and synthetic introns may provide additional tools for increasing transgene expression in plants.
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Abbreviations
- 35S:
-
Cauliflower mosaic virus 35S
- 5′ UTR:
-
5′ Untranslated region
- Gmubi:
-
Glycine max polyubiquitin
- MCS:
-
Multiple cloning site
- MS:
-
Murashige and Skoog
- OMS:
-
MS medium containing no plant growth regulators
- pFLEV:
-
Finer laboratory expression vector
- SINC:
-
Synthetic intron cassette
- TIF:
-
Tetrameric intron fragment
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Acknowledgements
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 work was partly supported by an OARDC Director’s Associateship Award to NZ in 2011–2012. 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.
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Grant, T.N.L., De La Torre, C.M., Zhang, N. et al. Synthetic introns help identify sequences in the 5′ UTR intron of the Glycine max polyubiquitin (Gmubi) promoter that give increased promoter activity. Planta 245, 849–860 (2017). https://doi.org/10.1007/s00425-016-2646-8
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DOI: https://doi.org/10.1007/s00425-016-2646-8