Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Dec;16(12):1971-1982.
doi: 10.1111/pbi.12931. Epub 2018 May 21.

Chimeric 3' flanking regions strongly enhance gene expression in plants

Andrew G Diamos  1 , Hugh S Mason  1
Affiliations

Chimeric 3' flanking regions strongly enhance gene expression in plants

Andrew G Diamos et al. Plant Biotechnol J. 2018 Dec.

Abstract

Plants represent a promising platform for the highly scalable production of recombinant proteins. Previously, we identified the tobacco extensin terminator lacking its intron as an element that reduced transcript read-through and improved recombinant protein production in a plant-based system. In this study, we systematically compared nonreplicating plant expression vectors containing over 20 commonly used or newly identified terminators from diverse sources. We found that eight gene terminators enhance reporter gene expression significantly more than the commonly used 35S and NOS terminators. The intronless extensin terminator provided a 13.6-fold increase compared with the NOS terminator. Combining terminators in tandem produced large synergistic effects, with many combinations providing a >25-fold increase in expression. Addition of the tobacco Rb7 or TM6 matrix attachment region (MAR) strongly enhanced protein production when added to most terminators, with the Rb7 MAR providing the greatest enhancement. Using deletion analysis, the full activity of the 1193 bp Rb7 MAR was found to require only a 463-bp region at its 3' end. Combined terminators and MAR together provided a >60-fold increase compared with the NOS terminator alone. These combinations were then placed in a replicating geminiviral vector, providing a total of >150-fold enhancement over the original NOS vector, corresponding to an estimated yield of 3-5 g recombinant protein per kg leaf fresh weight or around 50% of the leaf total soluble protein. These results demonstrate the importance of 3' flanking regions in optimizing gene expression and show great potential for 3' flanking regions to improve DNA-based recombinant protein production systems.

Keywords: 3’ UTR; biopharmaceutical; geminiviral vector; gene terminator; matrix attachment region; recombinant protein; transient expression.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Evaluation of diverse 3’ UTRs on GFP production. (a) Generalized schematic representation of the T‐DNA region of the vectors used in this study. RB and LB, the right and left borders of the T‐DNA region; NPTII, kanamycin resistance cassette; P35S, 35S promoter from cauliflower mosaic virus; TMV, 5’ UTR from tobacco mosaic virus; 3’ UTR is either a single terminator, double terminator, matrix attachment region or combination of these elements as described in each experiment. (b) Nonreplicating vectors containing 3’ UTRs inserted downstream from the GFP gene were agroinfiltrated into N. benthamiana leaves. Leaves were photographed at 5 DPI under UV illumination (365 nm). Images are representative of 3–4 independently infiltrated leaves. Agroinfiltrated leaves were harvested between 4 and 5 DPI and extracts were analysed by SDS‐PAGE followed by observation under UV illumination (365 nm) and Coomassie staining. GFP band intensity was quantified using ImageJ software, using native plant protein bands as a loading control. Data are means ± SE of 3–4 independently infiltrated leaves. Abbreviations: EU, intronless tobacco extensin 3’ UTR; IEU, intron‐containing tobacco extensin 3’ UTR; NbACT3, N. benthamiana actin 3’ UTR; NbHSP; N. benthamiana 18.8 kDa class II heat‐shock protein 3’ UTR; pinII, potato proteinase inhibitor II 3’ UTR; rbcS, pea rubisco small subunit 3’ UTR; SIR, short intergenic region of bean yellow dwarf virus; BDB, bean dwarf mosaic virus DNA B nuclear shuttle protein 3’ UTR; Rep, bean dwarf mosaic virus rep gene 3’ UTR; RepA, bean dwarf mosaic virus repA gene 3’ UTR; AtHSP, A. thaliana heat‐shock protein 3’ UTR; 35S, cauliflower mosaic virus 35S 3’ UTR; NOS, agrobacterium nopaline synthase 3’ UTR.
Figure 2
Figure 2
Double terminators strongly enhance GFP gene expression. Nonreplicating vectors containing different double terminators downstream from the GFP gene were agroinfiltrated into N. benthamiana leaves and analysed for GFP production at 5 DPI. Red bars indicate double terminators. Data are means ± SE of 3–4 independently infiltrated leaves. Abbreviations: BYDV, barley yellow dwarf virus 3’ UTR; PEMV, pea enation mosaic virus 3’ UTR; TNVD, tobacco necrosis virus‐D 3’ UTR; TMV, tobacco mosaic virus 3’ UTR; LIR, long intergenic region from bean yellow dwarf virus. For all other abbreviations, see Figure 1 legend.
Figure 3
Figure 3
MARs strongly enhance GFP expression. Nonreplicating GFP vectors containing either the tobacco Rb7 or tobacco TM6 MAR sequences inserted 3’ of the gene terminator were agroinfiltrated into the leaves of N. benthamiana and evaluated for GFP production at 5 DPI. Data are means ± SE of 3–4 independently infiltrated leaves. ‘EU + Control’ indicates DNA sequence obtained from an inverted region of the Norwalk virus capsid protein coding sequence was inserted 3’ of the EU gene terminator in place of the Rb7 MAR.
Figure 4
Figure 4
Combined 3’ UTRs strongly enhance GFP expression. Nonreplicating GFP vectors with combined terminators were created, agroinfiltrated into the leaves of N. benthamiana and evaluated for GFP production at 5 DPI. Data are means ± SE of 3–4 independently infiltrated leaves. Green bars indicate double terminators combined with Rb7 MAR; purple bars indicate double terminators combined with TM6 MAR; blue bars indicate single terminators combined with Rb7 MAR.
Figure 5
Figure 5
Evaluation of combined 3’ UTRs in replicating vectors. Replicating vectors containing elements of bean yellow dwarf virus (Diamos et al., 2016) were constructed with various combined 3’ flanking regions, agroinfiltrated into the leaves of N. benthamiana and evaluated for GFP production. (a) Data are means ± SE of 3–4 independently infiltrated leaves. ‘(R)’ indicates replicating geminiviral vector. (b) SDS‐PAGE gels showing GFP expression with the indicated constructs. RbcL; the large subunit of Rubisco .***P < 0.01
Figure 6
Figure 6
Comparison of selected 3’ UTRs expressing DsRed. Nonreplicating vectors were constructed with single, double or MAR‐containing terminators downstream from the DsRed gene and agroinfiltrated into the leaves of N. benthamiana. DsRed production was evaluated at 5 DPI by SDS‐PAGE and UV fluorescence. Data are means ± SE of 3–4 independently infiltrated leaves.
Figure 7
Figure 7
Comparison of selected 3’ UTRs in tobacco and lettuce. Nonreplicating vectors were constructed with single, double or MAR‐containing terminators downstream from the GFP gene and agroinfiltrated into the leaves of either tobacco (a) or lettuce (b) plants. GFP production was evaluated at 5 DPI by UV fluorescence and SDS‐PAGE. Data are means ± SE of 3–4 independently infiltrated leaves.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Allen, E. and Howell, M.D. (2010) miRNAs in the biogenesis of trans‐acting siRNAs in higher plants. Semin. Cell Dev. Biol. 21, 798–804. - PubMed
    1. Allen, G.C. , Hall, G. , Michalowski, S. , Newman, W. , Spiker, S. , Weissinger, A.K. and Thompson, W.F. (1996) High‐leve1 transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco. Plant Cell 8, 899–913. - PMC - PubMed
    1. Andersen, P.K. , Lykke‐Andersen, S. and Jensen, T.H. (2012) Promoter‐proximal polyadenylation sites reduce transcription activity. Genes Dev. 26, 2169–2179. - PMC - PubMed
    1. Baeg, K. , Iwakawa, H.O. and Tomari, Y. (2017) The poly(A) tail blocks RDR6 from converting self mRNAs into substrates for gene silencing. Nat. Plants 3, 17036. - PubMed
    1. Béclin, C. , Boutet, S. , Waterhouse, P. and Vaucheret, H. (2002) A branched pathway for transgene‐induced RNA silencing in plants. Curr. Biol. 12, 684–688. - PubMed

Publication types

MeSH terms

Substances