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Plant Molecular Biology

, Volume 96, Issue 4–5, pp 429–443 | Cite as

An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves

  • Sun Hee Rosenthal
  • Andrew G. Diamos
  • Hugh S. Mason
Article
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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.

Keywords

Polyadenylation Plant based expression Tobacco extensin terminator Read-through transcription Polypurine sequence Intron 

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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Notes

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.

Author contributions

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.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Supplementary material

11103_2018_708_MOESM1_ESM.tif (102 kb)
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)
11103_2018_708_MOESM2_ESM.tif (2.6 mb)
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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Authors and Affiliations

  1. 1.The Biodesign Institute, Center for Immunotherapy, Vaccines, and Virotherapy, School of Life SciencesArizona State UniversityTempeUSA

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