Efficient expression of a heterologous gene in plants depends on the nucleotide composition of mRNA’s 5'-region

Abstract

The contribution of nucleotide composition of mRNA 5'-region to the efficiency of expression at transcriptional and translational levels was studied in transgenic tobacco plants (Nicotiana tabacum L., cultivar Petit Havana) using a thermostable lichenase reporter gene. Synthetic sequence that contains CG-rich motifs, typical for 5'-region of plant genes, identified in silico, was constructed. Transgenic plant lines of N. tabacum were obtained; they contain thermostable lichenase reporter gene that is under control of the constitutive 35S RNA CaMV promoter and additional regulatory element: synthetic CG-rich sequence, which functions as the 5'-UTR (untranslated region) of the reporter gene mRNA or as 5'-region of the hybrid gene coding sequence, wherein the synthetic sequence is fused with the sequence of the reporter gene in its reading frame. Results of the comparative analysis of mRNA and protein levels in the obtained lines of transgenic plants showed that the synthetic CG-rich sequence significantly increases the level of transcription of the reporter gene and appear to have no negative effect on the efficiency of reporter mRNA translation, which may be due to the peculiarities of its nucleotide composition and structure, namely, due to the presence of motifs that are specific for 5'-regions of plant genes, as well as due to the properties of the secondary structure-the absence of hairpin structures with a high energy of formation. It was experimentally confirmed for the first time that the 5'-region of the genes with a high content of CpG dinucleotides can help to increase the transcription level of genes in plants.

Cis-regulatory elements used to control gene expression in plants

“…Furthermore, correct transcript binding to the ribosomal subunits significantly depends on the overall nature of 5′UTR regions. Due to economic considerations, forming of active translation complex only takes place in the presence of all translation factors (Tyurin et al 2016).…”

A High Throughput Assay of Lichenase Activity with Congo Red Dye in Plants

“…Moreover, CR does not require any specific binding sites in polysaccharides (β-glucans) [12]. According to earlier experimental data, CR binds to unhydrolyzed lichenan [22,23,24], which suggests that CR can form a complex with unhydrolyzed lichenan (β-1,3-1,4-glucan) and the amount of this complex is fluorometrically quantifiable. To confirm this assumption, we first recorded the fluorescence spectra of CR water solution (71.77 µM or 0.005%).…”

Computational and Experimental Tools to Monitor the Changes in Translation Efficiency of Plant mRNA on a Genome-Wide Scale: Advantages, Limitations, and Solutions

“…For studies of this type, expression cassettes are constructed that carry the reporter gene sequence with the expression controlled by a particular regulatory region or sequence selected by researcher (Figure 7). Researchers have at their disposal several reporter systems that have proved their efficiency in the studies of potential regulatory sequences or the nucleotide contexts that modulate translation in plant systems, in particular, β-glucuronidase (GUS); different variants of fluorescent proteins (for example, GFP and RFP); luciferases (Renilla luciferase, RLuc, and firefly luciferase, FLuc); and thermostable lichenase (LicBM) [51,52]. Commercial substrates and kits as well as the quantification methods for assessing the corresponding protein products are available for these reporter systems.…”

“…When experimentally confirming the role of the full-sized 5’UTRs in transcripts, these sequences are cloned upstream of the 5’ region of a reporter gene (Figure 7). Quantitative estimate of the reporter gene protein product when using different target 5’UTRs versus the known translational enhancers of various origins makes it possible to assess their contribution to the translation efficiency [51]. For example, A. thaliana mRNAs that are stably translated under any growth and environmental conditions have been found by polysome profiling.…”

“…When using the methods involving both stable and transient expressions, the choice of an adequate control is of a paramount importance to ascertain that the change in expression of the reporter protein is actually associated with the change in translation (rather than with transcription, protein stability, and so on) [4,51].…”