Isolation And Functional Analysis Of SHSP Genes In Sweet Pepper Under Temperature Stress

All organisms respond to elevated temperatures with production of a defined set of proteins called heat shock protein. The small heat shock proteins (sHSP) are ubiquitous in nature, but unusually abundant and diverse in higher plants as oppose to other eukaryotes. The sHSPs range in size from 17 to 30 kDa and share a conserved C-terminal domain. In higher plants six nuclear gene families encoding sHSPs have been defined. Each gene family encodes proteins founding a distinct cellular compartment, including the cytocol, chloroplast, ER and mitochondrion. In general, the sHSPs are not found in normal vegetative tissues, but accumulate to high levels in response to heat stress. Recently, a salient process involved in the production of sHSP induced by heat shock and the relationship between sHSP and thermotolerance was proposed. However, little is known about the production of sHSP induced by chilling stress and relationship between sHSP and chiliing tolerance. In this study, we presented the characterization of two sHSP genes from sweet pepper. Sequence analysis and RNA hybridization revealed that one of the two genes is most likely a chloroplast sHSP and the other is cytosolic class ⅠsHSP gene. The expression pattern and functional analysis showed that expression of these two genes was induced by heat stress. Interestingly, expression of these two genes was also induced by chilling stress, and sHSP play a role in protection of the proteins in tobacco and bacteria. The results are as follows: 1. Two degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from sweet pepper leaves on the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. The full length of the cDNA was isolated by 5'-RACE and 3'-RACE. The clone contains 917 nucleotides with an open reading frame (ORF) of 708 bp comprising 235 amino acid residues with the predicted molecular mass of 26 kD. The deduced amino acid sequence showed high identities with chloroplast sHSP from tobacco, Arabidopsis and maize. This indicated that the full length of the cDNA encoded a chloroplast sHSP, which was designed as CaHSP26. The phylogenetic analysis of various chloroplast sHSP indicated that tobacco, sweet pepper and Arabidopsis share one cluster. The deduced protein sequence exhibits a high degree of conservation in three consensus regions. Consensus region Ⅲknown as the "methionine rich domain", is the most conserved region within the predicted amino acid sequences of the chloroplast sHSP in several species of plants. A degenerate primer was designed to amplify specific DNA fragment using cDNA prepared from sweet pepper leaves on the homologous sequences from other plants. The 3' fragment of interested cDNA was obtained by RT-PCR. The full length of the cDNA was isolated by 5'-RACE. The clone contains 779 nucleotides with an open reading frame (ORF) of 480 bp comprising 159 amino acid residues with the predicted molecular mass of 18 kD. The deduced amino acid sequence of the gene showed high identities with cytosolic sHSP from tobacco, tomato and Arabidopsis. This indicated that the full length of the cDNA encoded a cytosolic sHSP, which was designed as CaHSP18. The phylogenetic analysis of various chloroplast sHSP indicated that sweet pepper, tobacco and maize share one cluster. The deduced protein sequence exhibits a high degree of conservation in two consensus regions near the carboxyl-terminal end of the protein. 2. Expression of CaHSP26 was obviously induced by high temperature (42℃) in all organs with the highest level in leaves. But no transcript was observed in any of the organs at 25℃. Transcripts were gradually enhanced after treatment for 3 h. But the transcript level declined in sweet pepper leaves kept at 42℃for 5 h. Sweet pepper plants were treated at 42℃for 1 h, and then recovered at 25℃or 4℃. The CaHSP26 transcripts survived longer at...