The HSP Terminator of Arabidopsis thaliana Induces a High Level of Miraculin Accumulation in Transgenic Tomatoes

Abstract

High-level accumulation of the target recombinant protein is a significant issue in heterologous protein expression using transgenic plants. Miraculin, a taste-modifying protein, was accumulated in transgenic tomatoes using an expression cassette in which the miraculin gene was expressed by the cauliflower mosaic virus (CaMV) 35S promoter and the heat shock protein (HSP) terminator (MIR-HSP). The HSP terminator was derived from heat shock protein 18.2 in Arabidopsis thaliana . Using this HSP-containing cassette, the miraculin concentration in T0 transgenic tomato lines was 1.4-13.9% of the total soluble protein (TSP), and that in the T1 transgenic tomato line homozygous for the miraculin gene reached 17.1% of the TSP. The accumulation level of the target protein was comparable to levels observed with chloroplast transformation. The high-level accumulation of miraculin in T0 transgenic tomato lines achieved by the HSP terminator was maintained in the successive T1 generation, demonstrating the genetic stability of this accumulation system.

Compatibility of translational enhancers with various plant species

“…For example, in order to achieve high accumulation of the mRNA of a foreign gene which is stably integrated it into nuclear genomes, the use of transcriptional terminator derived from Arabidopsis thaliana heat shock protein 18.2 gene has been effectively used in Arabidopsis thaliana, rice, lettuce, and tomato (Hirai et al 2011;Matsui et al 2011;Nagaya et al 2010). Furthermore, an improved version of the terminator has been developed (Matsui et al 2014).…”

Transcriptional enhancement of a bacterial <i>choline oxidase A</i> gene by an <i>HSP</i> terminator improves the glycine betaine production and salinity stress tolerance of <i>Eucalyptus camaldulensis</i> trees

“…These results agreed with previous reports: CaMV 35S promoter-derived reporter gene transcription under control of an HSP terminator was reported to be 1.7-to 2.7-fold higher than that under the control of an NOS terminator in Arabidopsis protoplast cells (Nagaya et al 2010). And miraculin protein accumulation in fruits of transgenic tomatoes expressing the miraculin transgene under control of an HSP terminator was 2.5-to 10-fold higher than that in fruits of transgenic tomatoes expressing this transgene under the control of an NOS terminator (Hirai et al 2011). The consistency of these results suggests that transcriptional enhancement by HSP terminator technology was also applicable in the stable transgenic trees of E. camaldulensis.…”

“…The HSP terminator from Arabidopsis thaliana has been demonstrated to increase the transcript of target transgenes in Arabidopsis, tobacco, rice, tomato, and lettuce (Hirai et al 2011;Kurokawa et al 2013;Matsui et al 2011;Nagaya et al 2010). In this study, we prepared two kinds of transgenic E. camaldulensis trees harboring CaMV 35S promoter-driven codA expression cassettes with different transcriptional terminators, i.e., the HSP terminator and NOS terminator ( Figure 1A) (NEDO 2013).…”

“…The heat shock protein (HSP) terminator derived from Arabidopsis thaliana has been demonstrated to increase both the transcription and translation of target transgenes in Arabidopsis, tobacco, rice, tomato, and lettuce (Hirai et al 2011;Kurokawa et al 2013;Matsui et al 2011;Nagaya et al 2010). Given this, we expected that the HSP terminator would enhance the transcription of exogenous abiotic stress-tolerance gene(s), and abiotic stress tolerance in Eucalyptus plantation trees.…”

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

“…We previously reported that the terminator derived from the Arabidopsis thaliana heat shock protein 18.2 gene (HSP, At5g59720) (HSPT) increases gene expression; the HSPT increases mRNA levels of both transiently and stably expressed transgenes approximately 2-fold more than widely-used NOS (nopaline synthase) terminator (Nagaya et al 2010). The HSPT is functional in a variety of plant cells including rice, A. thaliana, tomato, and lettuce (Hirai et al 2011;Matsui et al 2011;Nagaya et al 2010). In this study, with the goal of further increasing the expression level of transgenes, we evaluated a longer version of HSPT.…”