Cloning And Expression Of A Small Heat Shock Protein Gene CaHSP24 From Pepper

Heat-shock proteins (HSPs) are induced during thermal stress in all organisms examined, ranging from bacteria to human, and appear to be involved in thermoprotection; HSPs are usually divided into the high-molecular-weight (HMW) proteins of more than 30 kDa, and the low-molecular-weight (LMW) proteins of about 17 to 28 kDa. In contrast to animal systems, plants synthesize more LMW HSP than HMW HSP. Capsicum annuum L. is very sensitive to high temperature, but its responses to high temperature, especially with regard to sHSPs, have seldom been studied. In this study, we cloned a sHSP gene CaHSP24 from pepper and analyzed its expression patterns under heat stress and other abiotic stresses like salinity, heavy metal, cold, and oxidative stress. We also constructed a prokaryotic expression vector for CaHSP24 to study its effect on the viability of Escherichia coli under heat and cold stress. And we have constructed plant expression vector for CaHSP24, for the further studying of it's biological function by transgenic method. The main results of our research are as follows:1.The sequence of a small heat shock protein (sHSP) gene CaHSP24 was obtained by homology-based candidate gene method and rapid amplification of cDNA ends (RACE) from a heat resistant cultivar of pepper B35. The cDNA sequence of this gene is 920 bp in size (GenBank: HM132040) and contains an open reading frame (ORF) of 636 bp, which was predicted to encode a protein with 211 amino acid residues. The Blast alignment and phylogenic analysis of amino acid sequences showed that CaHSP24 was quite similar to mitochondrial sHSPs from other plants like zea mays, Arabidopsis, tomato. The similarity between CaHSP24 and LeHSP23.8 which is from tomato is as high as 88.07%, but CaHSP24 was distantly related to sHSPs of pepper cytoplasm and chloroplast.2. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) showed that CaHSP24 was hardly detectable at 32°C but accumulated markedly at 40°C. The gene was expressed 0.5 h after exposure to heat stress and the expression reached a peak in 1.5 h in the heat-resistant cultivar B35, while for the heat-sensitive cultivar B6, the expression level reached the peak in 1 h; but the expression level in cultivar B35 was higher than that in cultivar B6. The gene was also expressed weakly under salinity, heavy metal, low temperature, and oxidative stresses; but the expression levels under these conditions were remarkably lower than those under heat stress.3. We constructed a prokaryotic expression vector for CaHSP24, SDS-PAGE showed that recombinant CaHSP24 (27.5 kD) was expressed in pET28a-CaHSP24 cells 0.5 h after 1mmol/L IPTG-mediated induction, maximal expression was noted 2~5 h after induction, heterologous expression of CaHSP24 could enhance the viability of E. coli under heat and cold stress.4. We constructed the sense and anti-sense plant expression vector for CaHSP24, for the further studying of it's biological function by transgenic method.