Search NASA⌕ Search
Home/Archive/DOE OSTI

DOE OSTI · 3364800

Data for A Fluorescence-Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants

Haas, Benjamin [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:0000000234414151)·Saif, Faaiza [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA]·Bolger, Emily [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA]·Doran, Lynn [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)]·Rosales, Emma [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA]·Kim, Abigail [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA]·Burgess, Steven [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:0000000323537794)·Long, Stephen [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:0000000285017164)
View original source ↗Read source document ↗Export metadata ↓

Also available from

Abstract

Scripts for the manuscript "A fluorescence-based transient expression assay for the analysis of upstream open reading frames in plant" by Haas et al. Upstream open reading frames (uORFs) are regulatory elements present in the 5′ leaders of mRNA that can significantly impact downstream gene expression in eukaryotes. In crop engineering, editing of uORFs can provide an avenue to upregulate expression of native genes without the need to add persistent transgenic copies. Even with genome- wide methods to identify translated uORFs such as ribosome profiling, their functional characterization depends on validation through reporter gene assays and mutagenesis studies. Current screening methods for plants use luciferases or protoplasts to measure differential gene expression between wild- type and mutated transcript leaders, which requires tissue processing and/or substrate addition. Here, we present a time- and cost- efficient alternative to investigate transcript leaders by co- expression of two fluorescent proteins in Nicotiana benthamiana leaf tissue and test our assay on genes involved in photoprotection, editing of which could provide a pathway to increase CO2 assimilation during sun–shade transitions.

Explore related subjects

Gene EditingPhotosynthesisPlant TransformationTransient Expression

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Haas, Benjamin [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:0000000234414151), Saif, Faaiza [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA], Bolger, Emily [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA], Doran, Lynn [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)], Rosales, Emma [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA], Kim, Abigail [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA], Burgess, Steven [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:0000000323537794), Long, Stephen [Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)] (ORCID:0000000285017164). 2026-05-06. Data for A Fluorescence-Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants. https://doi.org/10.13012/b2idb-0009291_v1

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related reports

DOE OSTI2026

Data for Development, Optimization, and Application of an Episomal Plasmid System for Rhodotorula toruloides

Rhodotorula toruloides is an emerging oleaginous yeast with strong potential as a microbial cell factory for the production of acetyl-CoA-derived bioproducts. However, engineering of this organism has been limited by the absence of a functional episomal plasmid system, a foundational genetic tool for rapid gene expression, pathway testing, and CRISPR-based genome engineering. Here, we report the first episomal plasmid system for R. toruloides . Through systematic screening of candidate autonomously replicating sequences (ARSs) from diverse sources, we identified multiple functional ARS elements and selected C63F4, a fragment derived from Contig 63 of R. toruloides CBS14, because of its stable performance. The resulting pC63F4 plasmid was maintained episomally, supported GFP reporter expression, exhibited a copy number of 2.39 ± 0.13, and showed good stability during long term cultivation. To overcome poor transformation efficiency, we developed a Cre-loxP-mediated in vivo re-circularization strategy that enabled reliable delivery of the episomal plasmid. Using this improved system, we demonstrated functional episomal expression of metabolic engineering genes and multi-gene pathways for the production of triacetic acid lactone, fatty alcohols, and limonene. Finally, we leveraged this platform to establish a redesigned CRISPR system that enables seamless genome editing in R. toruloides for the first time, while also simplifying marker recycling. Together, this work establishes a long-needed episomal plasmid platform and associated CRISPR toolkit that will accelerate metabolic engineering, synthetic biology, and fundamental studies in R. toruloides .

Gene Editing↗
DOE OSTI2026

Data for Miscanthus giganteus Biolistic Transformation Using the Visible RUBY Red Marker Gene to Monitor Transformation Efficiency

Miscanthus × giganteus ( M × g ) is a high- yielding perennial C4 bioenergy crop, but genetic improvement by breeding is constrained by triploid sterility and clonal propagation. Improving genetic transformation methods for M × g would provide opportunities for advantageous trait introgression. Use of an easy to phenotype reporter gene is a promising strategy to improve transformation processes and efficiency. This study presents an efficient novel method for biolistic transformation of inflorescence- derived callus in M × g and demonstrates its efficacy using RUBY, a betalain-based noninvasive reporter that is visible throughout the transformation process. RUBY expression ( Zea mays codon optimized) was visible from callus stage through plantlet development into maturity. RUBY expressing independently transformed plants were confirmed by hygromycin phosphotransferase ELISA and by genomic PCR demonstrating that the RUBY phenotype is sufficient for screening transformants. The Zea mays codon optimized hygromycin selection marker was driven by previously established promoters for Miscanthus, ZmUBI and 2×35S, while the RUBY gene expression was controlled by a known Zea mays C4 promoter, Brachypodium UBI10, newly employed in Miscanthus. The construct containing the 2×35S promoter for hygromycin had a 15.1% transformation efficiency while the ZmUBI promoter had a 20.5% transformation efficiency. This study provides a novel, highly efficient protocol for successful biolistic transformation of M × g for stable expression. This study also demonstrates that RUBY expression can be used as a convenient and powerful monitor of transformation in ongoing and future work to engineer M × g into an improved bioproduct feedstock. **NOTE: in "TableS2_ProtocolComparison.csv", the data from row 665 to 971 should be removed.

Gene Editing↗