An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves
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.
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.
KeywordsPolyadenylation Plant based expression Tobacco extensin terminator Read-through transcription Polypurine sequence Intron
Auxiliary downstream elements
Cauliflower mosaic virus
Cleavage stimulating factor
Days post infiltration
Far upstream element
Flowering Locus C
Green fluorescent protein
Heat shock protein
Hydroxy proline-rich glycoprotein
Near upstream element
Norwalk virus capsid protein
Polyadenylation specificity factor
Post transcriptional gene silencing
RNA polymerase II
RNA-dependent RNA polymerase 6
Tobacco etch virus
Vegetative storage protein
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.
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.
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