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Updated: Jul 17, 2026
12:02
Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun
Published on: April 18, 2014
Chimeric 3' flanking regions strongly enhance gene expression in plants
Andrew G Diamos1, Hugh S Mason1
1Center for Immunotherapy, Vaccines and Virotherapy, Biodesign Institute at ASU, and School of Life Sciences, Arizona State University, Tempe, AZ, USA.
Plant Biotechnology Journal
|April 12, 2018
Summary
Optimizing plant-based protein production, researchers found that specific gene terminators and matrix attachment regions (MARs) significantly boost recombinant protein yields. Combining these elements achieved over 150-fold enhancement, demonstrating the potential of 3' flanking regions for scalable biomanufacturing.
Area of Science:
- Biotechnology
- Molecular Biology
- Plant Science
Background:
- Plants offer a scalable platform for recombinant protein production.
- The tobacco extensin terminator (without its intron) was previously identified to improve protein yields by reducing read-through.
- Optimization of 3' flanking regions is crucial for enhancing gene expression in plant systems.
Purpose of the Study:
- To systematically compare various gene terminators and matrix attachment regions (MARs) for their efficacy in enhancing recombinant protein production in plants.
- To identify optimal combinations of terminators and MARs for maximizing protein yield.
- To evaluate the performance of these optimized elements in a replicating viral vector.
Main Methods:
- Systematic comparison of over 20 plant gene terminators using non-replicating expression vectors.
- Evaluation of matrix attachment regions (MARs), including tobacco Rb7 and TM6, for their enhancement effects.
- Tandem combination of terminators and MARs to assess synergistic effects.
- Deletion analysis of the Rb7 MAR to identify essential regions for activity.
- Integration of optimized elements into a replicating geminiviral vector for high-level expression.
Main Results:
- Eight gene terminators significantly outperformed the commonly used 35S and NOS terminators.
- The intronless extensin terminator showed a 13.6-fold increase compared to the NOS terminator.
- Tandem terminator combinations yielded over a 25-fold increase in expression.
- Addition of MARs, particularly Rb7 MAR, strongly enhanced protein production.
- Combined terminators and MARs achieved over a 60-fold increase.
- In a replicating viral vector, total enhancement exceeded 150-fold, yielding 3-5g/kg fresh weight or ~50% of total soluble protein.
Conclusions:
- 3' flanking regions, including specific terminators and MARs, are critical for optimizing gene expression in plants.
- Combinatorial strategies using multiple terminators and MARs can lead to substantial synergistic increases in recombinant protein production.
- These optimized elements demonstrate significant potential for improving DNA-based biomanufacturing systems in plants, achieving high yields suitable for industrial applications.
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