Table_2_Design and Analysis of Native Photorespiration Gene Motifs of Promoter Untranslated Region Combinations Under Short Term Abiotic Stress Conditions.DOCX

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.

量化性状通常受单个基因控制的情况极为罕见，这使得多基因改造成为现代合成生物学方法中不可或缺的一环。然而，缺乏用于稳健地同时表达多个核转基基因的独特基因调控元件（GREs）成为了阻碍植物复杂途径工程化的主要瓶颈。在本研究中，我们对比了一系列经过充分记录的启动子和非翻译区（UTR）序列的转录效率。通过结合烟草的瞬时表达和双荧光素酶试验，我们考察了GREs的强度。此外，我们通过结合表现最佳的启动子-UTR序列，创建了具有更高转录效率的新GREs系列。我们还测试了高温和高照度对这些GREs有效性的影响。尽管组成型启动子确保了转基基因的稳健表达，但它们缺乏原生启动子所展现的空间和时间调控。在此，我们提出了一项基于三个关键光呼吸基因顺式调控元件的合成启动子特征化的原理性研究。这一保守的生化过程在高温、低二氧化碳和高照度应激条件下通常会增加，导致固定二氧化碳的约25%损失。为了选择参与光呼吸的应激响应顺式调控元件，我们利用PlantPAN3.0和AthaMap分析了两个叶绿体转运蛋白（AtPLGG1和AtBASS6）以及一个关键质体酶AtPGLP的启动子。我们的结果表明，这些基序在调节PLGG1、BASS6和PGLP对高温和高强度光照应激的反应中发挥着关键作用。这些发现不仅将促进光呼吸的代谢和遗传工程，还将成为相关合成生物学方法的有力工具。