Abstract
Key message
We have found interesting features of a plant gene (extensin) 3′ flanking region, including extremely efficient polyadenylation which greatly improves transient expression of transgenes when an intron is removed. Its use will greatly benefit studies of gene expression in plants, research in molecular biology, and applications for recombinant proteins.
Abstract
Plants are a promising platform for the production of recombinant proteins. To express high-value proteins in plants efficiently, the optimization of expression cassettes using appropriate regulatory sequences is critical. Here, we characterize the activity of the tobacco extensin (Ext) gene terminator by transient expression in Nicotiana benthamiana, tobacco, and lettuce. Ext is a member of the hydroxyproline-rich glycoprotein (HRGP) superfamily and constitutes the major protein component of cell walls. The present study demonstrates that the Ext terminator with its native intron removed increased transient gene expression up to 13.5-fold compared to previously established terminators. The enhanced transgene expression was correlated with increased mRNA accumulation and reduced levels of read-through transcripts, which could impair gene expression. Analysis of transcript 3′-ends found that the majority of polyadenylated transcripts were cleaved at a YA dinucleotide downstream from a canonical AAUAAA motif and a UG-rich region, both of which were found to be highly conserved among related extensin terminators. Deletion of either of these regions eliminated most of the activity of the terminator. Additionally, a 45 nt polypurine sequence ~ 175 nt upstream from the polyadenylation sites was found to also be necessary for the enhanced expression. We conclude that the use of Ext terminator has great potential to benefit the production of recombinant proteins in plants.
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Abbreviations
- AG:
-
Arabidopsis agamous
- auxDSE:
-
Auxiliary downstream elements
- CaMV:
-
Cauliflower mosaic virus
- CE:
-
Cleavage element
- CF:
-
Cleavage factors
- CstF:
-
Cleavage stimulating factor
- DPI:
-
Days post infiltration
- DSE:
-
Downstream element
- Ext:
-
Extensin
- FUE:
-
Far upstream element
- FLC:
-
Flowering Locus C
- GFP:
-
Green fluorescent protein
- HSP:
-
Heat shock protein
- HRGP:
-
Hydroxy proline-rich glycoprotein
- IME:
-
Intron-mediated enhancement
- NUE:
-
Near upstream element
- NOS:
-
Nopaline synthase
- NVCP:
-
Norwalk virus capsid protein
- OCS:
-
Octopine synthase
- poly(A):
-
Polyadenylation
- PAP:
-
Poly(A) polymerase
- CPSF:
-
Polyadenylation specificity factor
- PPS:
-
Polypurine sequence
- PTGS:
-
Post transcriptional gene silencing
- RNAPII:
-
RNA polymerase II
- RDR6:
-
RNA-dependent RNA polymerase 6
- STK:
-
Seedstick
- TEV:
-
Tobacco etch virus
- UTR:
-
Untranslated region
- USE:
-
Upstream element
- VSP:
-
Vegetative storage protein
- GUS:
-
β-Glucuronidase
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Acknowledgements
This work was supported by Grant NIH-NIAID 1 U19 AI066332-01 to H.S.M. The authors wish to thank Charles Arntzen, Yung Chang, and Tsafrir Mor for helpful discussion and advice on the manuscript.
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SR and AD conceived the studies, conducted the experiments, analyzed the data and wrote the manuscript. HSM supervised the design of the study and data analysis and critically finalized the manuscript. The authors read and agreed to the final manuscript.
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Sun Hee Rosenthal and Andrew G. Diamos have contributed equally to this work.
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Fig. S1
The extensin terminator has strong activity in other plants. GFP constructs containing the extensin terminator with or without intron were agroinfiltrated into the leaves of (a) lettuce or (b) tobacco. At 4 DPI, leaf tissue was harvested and protein extracts were analyzed by SDS-PAGE followed by observation under UV illumination (365 nm) and by Coomassie staining. GFP band intensity was quantified using ImageJ software, using native plant proteins as a loading control. Columns represent means ± standard error of three independently infiltrated leaves. All leaves were infiltrated with construct pEU as an internal control for leaf and plant variability. (TIF 102 KB)
Fig. S2
Alignment of related extensin terminators. Nucleotide sequences of homologous extensin terminators were obtained from GenBank or the Sol Genomics Network and aligned with the tobacco extensin terminator using the Clustal Omega program. Conserved features identified in this study are highlighted. (TIF 2654 KB)
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Rosenthal, S.H., Diamos, A.G. & Mason, H.S. An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves. Plant Mol Biol 96, 429–443 (2018). https://doi.org/10.1007/s11103-018-0708-y
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DOI: https://doi.org/10.1007/s11103-018-0708-y