Carrot (DaucuscarotaL.) heat shock protein 70 gene (DcHsp70) confers tolerance to heat or cold stress in E. coli cells

Abstract

SummaryA heat shock protein 70 gene (DcHsp70) from carrot (Daucus carota L. ‘Mussangochon’) was isolated using PCR.The gene was 1,959 bp-long and the deduced amino acid sequence suggested that the encoded protein was composed of 652 amino acids (approx. 71 kDa). Sequence alignment revealed that the protein contained an ATPase domain at its N-terminus and a substrate-binding domain at its C-terminus. It also had the characteristic EEVD sequence for cytosolic localisation. Reverse transcription-PCR revealed the absence of DcHsp70 transcripts in non-stressed leaf tissue. However, heat or cold stress induced DcHsp70 gene expression. To examine the function of DcHsp70, the coding region of the gene was introduced into Escherichia coli BL21 (DE3) and the product was expressed following induction with isopropyl _-D-1-thiogalactopyranoside (IPTG). Under normal growth temperatures (37°C), the transformed E. coli cells showed similar growth rates to the vector-only control. However, under heat stress (50°C), the transformed E. coli cells displayed higher viability (35%) compared to the vector-only control (15%). Under cold stress (2°C), the transformed E. coli cells also displayed higher viability (71%) compared to the vector-only control (43%).Transformed E. coli cells had higher concentrations of soluble proteins under either stress condition compared to the vector-only control, suggesting that heterologously-expressed DcHSP70 protein functioned as a molecular chaperone to maintain the solubility of proteins. Adenosine triphosphate (ATP) further enhanced the molecular chaperone function of DcHSP70 under either stress condition. The DcHsp70 gene could be a candidate to produce transgenic crops with enhanced heat/cold stress tolerance.