Dataset Information

Identification of 5'-untranslated regions that function as effective translational enhancers in monocotyledonous plant cells using a novel method of genome-wide analysis.

ABSTRACT: High expression of a transgene is often necessary to produce useful substances in plants. The efficiency of mRNA translation is an important determinant of the level of transgene expression. In dicotyledonous plants, the 5'UTR of certain mRNAs act as translational enhancers, dramatically improving transgene expression levels. On the other hand, translation enhancers derived from dicotyledonous plants are not so much effective in monocotyledonous plants, which are important as industrial crops and as hosts for production of useful substances. In this study, we evaluated the polysome association on a large scale with high resolution for each 5'UTR variant from multiple transcription start site in normal and heat-stressed Oryza sativa suspension cultures. Translational enhancer candidates were selected from the resultant large-scale data set, and their enhancer activities were evaluated by transient expression assay. In this manner, we obtained several translational enhancers with significantly higher activities than previously reported enhancers. Their activities were confirmed in a different monocotyledonous plant, Secale cereale, and using a different reporter gene. In addition, enhancer activities of tested 5'UTRs were different between monocotyledonous and dicotyledonous plants, suggesting that the enhancer activities were not compatible between them. Overall, we demonstrate these useful 5'UTRs as enhancer sequence for transgene expression in monocotyledonous plants.

SUBMITTER: Yamasaki S

PROVIDER: S-EPMC6905215 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Publications

Identification of 5'-untranslated regions that function as effective translational enhancers in monocotyledonous plant cells using a novel method of genome-wide analysis.

Yamasaki Shotaro S Suzuki Atsunobu A Yamano Yasuaki Y Kawabe Harunori H Ueno Daishin D Demura Taku T Kato Ko K

Plant biotechnology (Tokyo, Japan) 20181201 4

High expression of a transgene is often necessary to produce useful substances in plants. The efficiency of mRNA translation is an important determinant of the level of transgene expression. In dicotyledonous plants, the 5'UTR of certain mRNAs act as translational enhancers, dramatically improving transgene expression levels. On the other hand, translation enhancers derived from dicotyledonous plants are not so much effective in monocotyledonous plants, which are important as industrial crops an ...[more]

PMID: 31892824

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Project description:BackgroundThe growing field of plant molecular farming relies on expression vectors that allow high yields of recombinant proteins to be produced through transient gene expression. While numerous expression vectors currently exist for this purpose, there are very few examples of systematic efforts to improve upon these. Moreover, the current generation of expression systems makes use of naturally-occurring regulatory elements, typically selected from plant viruses, to maximise yields. This study aims to use rational design to generate synthetic sequences that can rival existing ones.ResultsIn this work, we present the rational design of novel synthetic 5' and 3' untranslated regions (UTRs) which can be used in various combinations to modulate accumulation levels of transiently-expressed recombinant proteins. Using the pEAQ-HT expression vector as a point of comparison, we show that pre-existing expression systems can be improved by the deployment of rationally designed synthetic UTRs. Notably, we show that a suite of short, synthetic 5'UTRs behave as expression enhancers that outperform the HT 5'UTR present in the CPMV-HT expression system. Furthermore, we confirm the critical role played by the 3'UTR of cowpea mosaic virus RNA-2 in the performance of the CPMV-HT system. Finally, we use the knowledge obtained from these results to develop novel expression vectors (named pHRE and pHREAC) that equal or outperform pEAQ-HT in terms of recombinant protein yield. These new vectors are also domesticated for the use of certain Type IIS restriction enzymes, which allows for quicker cloning and straightforward assessment of different combinations of UTRs.ConclusionsWe have shown that it is possible to rationally design a suite of expression modulators in the form of synthetic UTRs. We have created novel expression vectors that allow very high levels of recombinant protein expression in a transient expression context. This will have important consequences for future efforts to develop ever-better plant transient overexpression vectors for research or industrial applications.

Dataset Information

Identification of 5'-untranslated regions that function as effective translational enhancers in monocotyledonous plant cells using a novel method of genome-wide analysis.

Publications

Identification of 5'-untranslated regions that function as effective translational enhancers in monocotyledonous plant cells using a novel method of genome-wide analysis.

Similar Datasets

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