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

Abstract

In order to achieve higher expression of a transgene, it is important to optimize not only the promoter, 5′-untranslated region (UTR), and localization signal, but also the transcriptional terminator. We previously reported that the terminator derived from the Arabidopsis thaliana heat shock protein 18.2 gene (HSPT) increases gene expression in various plant species. In this study, with the goal of further increasing the expression level of transgenes, we evaluated a longer version of HSPT, corresponding to the 878 bp downstream of the stop codon, in cultured tobacco cells. The longer version of HSPT increased the expression levels of various genes including Renilla reniformis luciferase, Photinus pyralis luciferase, horseradish (Armoracia rusticana) peroxidase, and the non-toxic B subunit of Stx2e, a candidate vaccine protein for pig edema disease. This effect was not observed in a transient expression system. This element represents a useful tool for expression of transgenes integrated into the nuclear genome in plant cells.

Plant 3’ Regulatory Regions From mRNA-Encoding Genes and Their Uses to Modulate Expression

“…Perhaps it is also due to APA events, allowing fine regulation of expression (Takagaki et al, 1996;Tang et al, 2002;Quesada et al, 2003;Simpson et al, 2003;Delaney et al, 2006;Dong et al, 2007;Ji et al, 2009;Tian and Manley, 2013). Matsui et al (2014) demonstrated that the use of a longer version of the HSP 3' regulatory region results in higher levels of expression compared to a smaller version. According to the authors, the high levels of expression achieved in the longer version occurred due to the presence of a matrix attachment region (MAR), ATrich DNA sequences that assist in the chromatin structural organization, being involved in transcriptional control (Abranches et al, 2005;Tetko et al, 2006).…”

Generation of fluorescent flowers exhibiting strong fluorescence by combination of fluorescent protein from marine plankton and recent genetic tools in <i>Torenia fournieri</i> Lind.

“…The resulting plasmid was digested with NsiI, followed by treatment with T4 DNA polymerase to fix the NsiI site and generate AtADH_5′UTR:CpYGFP:HSP-T. Furthermore, a SacI-EcoRI HSP-T878 terminator fragment was excised from AtADHFluc-HSPT878 (Matsui et al 2014) and inserted into the SacIEcoRI gap of AtADH_5′UTR:CpYGFP:HSP-T to generate AtADH_5′UTR:CpYGFP:HSP-T878. An XbaI-EcoRI fragment was excised from AtADH_5′UTR:CpYGFP:HSP-T878 and inserted into the SpeI-EcoRI gap of AtADH_5′UTR: CpYGFP:HSP-T878 to generate 2×CpYGFP.…”

“…A considerable number of Torenia plants transformed with this improved gene construct (1×CpYGFP) clearly exhibited strong fluorescence observable without using any image-enhancing equipment ( Figure 2D, 1×CpYGFP plants). To gain stronger fluorescence, HSP-T (250 bp) in 1×CpYGFP was replaced with its longer version HSP-T878 (Matsui et al 2014), and the expression cassette was tandemly triplicated (3×CpYGFP; Figure 1, lower). A significant number of Torenia plants transformed with this improved gene construct exhibited an excellent strong fluorescence never observed before ( Figure 2E, 3×CpYGFP plants).…”

“…Accumulation of mRNA with HSP-T increases more than two times compared with the common nopaline synthase (NOS) terminator (NOS-T), and it also properly functions in rice and tomato (Hirai et al 2011;Nagaya et al 2010). The latest improved version of 878-bp HSP-T (HSP-T878) containing a matrix attachment region (MAR) increased the mRNA levels 1.5-to 2-fold higher than the previous version of HSP-T (250 bp) in stable transgenic plants (Matsui et al 2014). On the other hand, the translational enhancer sequence Ω from tobacco mosaic virus (Gallie et al 1991;Sleat et al 1987) has long been used as a convenient tool for transgene expression.…”

Generation of brilliant green fluorescent petunia plants by using a new and potent fluorescent protein transgene

“…In the present study, we designed an expression cassette in which the coding sequence of eYGFPuv was driven by the cauliflower mosaic virus (CaMV) 35S promoter combined with the transcriptional terminator of the heat shock protein 18.2 (HSP) gene of A. thaliana (HSP-T878) 24 and the 5′ untranslated region (5′UTR) of cold-regulated 47 (COR47) in A. thaliana (COR47-5′UTR) 25 . HSP-878 and COR 47-5′UTR have been reported to increase the expression level of the transgene compared to other transcriptional terminators and translational enhancer, and high accumulation of FPs is expected.…”