High level expression of transgenes by use of 5^|^#8242;-untranslated region of the Arabidopsis thaliana arabinogalactan-protein 21 gene in dicotyledons

Abstract

For higher expression of a foreign gene in plant cells, it is important to optimize nucleotide sequences corresponding to 5′-untranslated region (5′-UTR), because it usually has great impacts on the expression of the gene mainly at the translational level. In this study, with an aim to nd useful 5′-UTRs, thirty nine 5′-UTRs derived from Arabidopsis thaliana genes were tested by transient expression of re y luciferase (Fluc), and that of A. thaliana arabinogalactanprotein 21 (AtAGP21) gene was selected for further analyses. Its activity was either equaling or surpassing that of known translational enhancer, A. thaliana alchol dehydrogenase (AtADH) 5′-UTR in dicotyledons, and was further improved by the optimizing sequence context of the initiating codon (−3 to −1 of AUG). Finally, we also found that the modi ed AtAGP21 5′-UTR was useful in recombinant expression of horseradish peroxidase (HRP) in tobacco cultured cells, and the yield was as much as 23 mg l −1 culture medium in seven days.

Overexpression of an extraplastidic β-amylase which accumulates in the radish taproot influences the starch content of Arabidopsis thaliana

“…The RsBAMY1 gene was inserted at the 3′ terminus of the 5′ untranslated region (5′UTR) of the Arabidopsis alcohol dehydrogenase gene to enhance the efficiency of translation (Sugio et al 2008;Matsui et al 2012). The KOD DNA Polymerase (Toyobo, Osaka, Japan) used for the PCR.…”

“…For strong expression of RsBAMY1, we used the sequence of cauliflower mosaic virus 35S promoter and the 5′UTR of the Arabidopsis alcohol dehydrogenase gene to enhance transcription and translation (Sugio et al 2008;Matsui et al 2012). An empty vector was transformed to generate a control line (a vector control, VC).…”

R-STEINER: Generation Method of 5'UTR for Increasing the Amount of Translated Proteins

“…As a solution, we focus on the area 5'UTR (5'-untranslated region). It is known that gene expression is affected by 5'UTR sequence [5], and some researchers have tried to discover 5'UTR sequences that increase the amount of translated proteins of certain genes [5], [6], [7], [8], [9]. However, real experiments done to discover the transla- s-nakamura@is.naist.jp tion enhancer-referring to the 5'UTR sequences which increase the amount of translated proteins-involve significant costs and require time and effort.…”

The longer version of Arabidopsis thaliana heat shock protein 18.2 gene terminator contributes to higher expression of stably integrated transgenes in cultured tobacco cells

“…In order to increase the accumulated levels of a recombinant protein, it is often necessary to optimize the expression cassette. At the translational level, the 5′-untranslated region (5′-UTR) plays an important role, because the nucleotides immediately upstream of the initiating AUG (−3 to −1) exert a considerable influence on translation initiation (Sugio et al 2010), and some 5′-UTRs function as translational enhancers in plant cells (Matsui et al 2012;Satoh et al 2004;Sugio et al 2008). At the posttranslational level, targeting the protein into a vesicular transport pathway often leads to increased accumulation (Yoshida et al 2004).…”

“…Horseradish (Armoracia rusticana) peroxidase (HRP) is used for a variety of detection procedures such as western blotting and enzyme-linked immunosorbent assay (ELISA). We previously reported high-level production of secreted HRP using BY2 cells (Matsui et al 2012). Here we used an HRP-expression construct in which HRP was fused to a variant of the 5′-UTR of Arabidopsis thaliana arabinogalactan-protein 21 (AtAGP21) containing a modification to the sequence context of the initiation codon (−3 to −1 bp before AUG).…”