[cDNA cloning and expression of a cytosolic small heat shock protein gene (CaHSP18) from Capsicum annuum]

Abstract

Full length 779 bp cytosolic clsss I sHSP cDNA was cloned from heat-shocked sweet pepper leaves using a PCR approach with degenerate primers designed from conserved motifs found in a number of plant cytosolic clsss I sHSP genes, named CaHSP18. Its accession number is AY284925. CaHSP18 was high identified with Nicotiana tabacum (Fig.1). A multi-gene family was found in sweet pepper genomic DNA by Southern-blot analysis (Fig.3). Northern blot revealed that the expression of CaHSP18 in sweet pepper was induced by heat treatment and was significantly different between different tissues (Fig.4). The transcription of CaHSP18 in leaves and stems were higher than those in roots. The expression of CaHSP18 could be detected after chilling treatment at 4 degrees C for 2 d. Recombinant CaHSP18 was overexpressed in Escherichia coli to study its possible function under heat and chilling stress. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that CaHSP18 was expressed in Escherichia coli (Fig.6). The growth of wild type and transformed cells was similar at 37 degrees C (Fig. 5). Upon transfer from 37 degrees C to 50 degrees C, a temperature known to cause cell autolysis, those cells that accumulated CaHSP18 showed improved viability compared with the control lines (Fig.7). To test the hypothesis that sHSPs may be involved in protection against chilling stress, the viability of recombinant cells at 4 degrees C was studied. The sweet pepper CaHSP18 significantly enhanced cell survivability (Fig.8). These results indicate that plant cytosolic clsss I sHSP have protective functions not only against heat stress but also against chilling stress.