Heat-inducible expression system for a foreign gene in cultured tobacco cells using the HSP18.2 promoter of Arabidopsis thaliana

Abstract

A system for the controlled expression of a foreign gene in cultured tobacco cells (Nicotiana tabacum, BY2) by temperature shift was constructed. A 925-base-pair (bp) DNA fragment containing the 5'-flanking region of a low-molecular-mass heat-shock protein gene (HSP18.2) of Arabidopsis thaliana was inserted upstream of the beta-glucuronidase reporter gene (GUS). The resulting HSP18.2-GUS construct was introduced into BY2 cells by electroporation or Agrobacterium-mediated transformation. Transient expression of the HSP18.2 promoter in protoplasts was very low regardless of the heat shock. Although expression of the HSP18.2-GUS chimeric gene in the stable transformants of BY2 was hardly detected in culture at 25 degrees C, the expression increased rapidly on the transcriptional level when the incubation temperature was shifted to 35-37 degrees C. The optimal temperature for heat-shock induction was 37 degrees C. After a 2-h incubation at 37 degrees C, GUS activity was about 1000-fold greater than that before heat shock. The amount of GUS mRNA was maximum 2 h after heat shock, and then decreased gradually.

Controlled Expression of Recombinant Proteins in Physcomitrella patens by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

“…Thus, in Nicotiana tabacum the promoter hsp17.6L from soybean can control antibiotic resistance in a temperature-dependent manner (Severin and Schoffl, 1990) as well as the expression of the FLP recombinase in maize (Lyznik et al, 1995) and Arabidopsis thaliana (Kilby et al, 2000). Similarly, the A. thaliana hsp18.2 promoter was successfully used as a heat-inducible expression system in tobacco BY2 cells (Yoshida et al, 1995), and to achieve heat-inducible RNAi expression in A. thaliana (Masclaux et al, 2004). Recently, using the soybean Gmhsp17.3B promoter, inducible insertional mutagenesis, based on Ac-Ds transposons, was developed in A. thaliana (Nishal et al, 2005).…”

A Tightly Regulated Genetic Selection System with Signaling-Active Alleles of Phytochrome B

“…The Arabidopsis HSP18.2 HS promoter has been widely used in plant biotechnology for tight temporal induction of target genes of interest (Takahashi et al, 1992;Yoshida et al, 1995;Matsuhara et al, 2000;Masclaux et al, 2004;Luo et al, 2008). In this regard, HSP18.2 expression is nearly undetectable in 4-to 7-d-old seedlings (Hu and Ma, 2006;Hu et al, 2009) as well as in most adult plant tissues maintained at/or below 25°C (Supplemental Fig.…”

Inducible Gene Expression Systems for Plants

“…The cisactivation is carried out by cloning the gene of interest downstream of the HSP18.2 promoter and by inducing the transgenic plant with heat shocks at 37 • C. In absence of heat stress, the HSP18.2 promoter is known to be repressed (36 3. Dexamethasone-containing lanolin paste.…”