Abstract
Quantitative traits are rarely controlled by a single gene, thereby making multi-gene transformation an indispensable component of modern synthetic biology approaches. However, the shortage of unique gene regulatory elements (GREs) for the robust simultaneous expression of multiple nuclear transgenes is a major bottleneck that impedes the engineering of complex pathways in plants. In this study, we compared the transcriptional efficacies of a comprehensive list of well-documented promoter and untranslated region (UTR) sequences side by side. The strength of GREs was examined by a dual-luciferase assay in conjunction with transient expression in tobacco. In addition, we created suites of new GREs with higher transcriptional efficacies by combining the best performing promoter-UTR sequences. We also tested the impact of elevated temperature and high irradiance on the effectiveness of these GREs. While constitutive promoters ensure robust expression of transgenes, they lack spatiotemporal regulations exhibited by native promoters. Here, we present a proof-of-principle study on the characterization of synthetic promoters based on cis-regulatory elements of three key photorespiratory genes. This conserved biochemical process normally increases under elevated temperature, low CO2, and high irradiance stress conditions and results in ∼25% loss in fixed CO2. To select stress-responsive cis-regulatory elements involved in photorespiration, we analyzed promoters of two chloroplast transporters (AtPLGG1 and AtBASS6) and a key plastidial enzyme, AtPGLP using PlantPAN3.0 and AthaMap. Our results suggest that these motifs play a critical role for PLGG1, BASS6, and PGLP in mediating response to elevated temperature and high-intensity light stress. These findings will not only enable the advancement of metabolic and genetic engineering of photorespiration but will also be instrumental in related synthetic biology approaches.
Highlights
The construction and expression of multiple genes simultaneously are necessary to engineer complex genetic circuits and regulatory networks underlying almost every metabolic, developmental, and signaling pathway in plants (Lozada et al, 2017; Zhang et al, 2020; Ma et al, 2021)
Even constitutive expression can be modulated by photoperiod, temperature and the developmental stage of the plants (Obertello et al, 2005; Boyko et al, 2010), we investigated the effect of elevated temperature on constitutive promoters and untranslated region (UTR) combinations
We developed a rapid screening method to test the strength and efficacies of several promoters and UTRs by employing a dual luciferase-based transient transcriptional activity assay in Nicotiana benthamiana
Summary
The construction and expression of multiple genes simultaneously are necessary to engineer complex genetic circuits and regulatory networks underlying almost every metabolic, developmental, and signaling pathway in plants (Lozada et al, 2017; Zhang et al, 2020; Ma et al, 2021). To facilitate such complex multigene engineering, the development of suites of unique. Plant engineering attempts relied on repetitive usage of similar GREs, thereby often negatively influencing plasmid stability (Peremarti et al, 2010) Such approaches have an increased probability of homology-based gene silencing (Oliveira et al, 2010). Transforming single vectors containing multiple expression cassettes has emerged as an efficient method for the simultaneous introduction of multiple genes
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