Molecular Characteristics, Prokaryotic Expression And Transcreptional Expression Analysis Of A Heat Shock Protein Gene CpHSP1 From Chimonanthus Praecox

The accumulation of heat shock proteins (HSPs) after exposure of cells or organisms to variety of environmental stresses including elevated temperatures, chilling temperatures, hormone,salinity,aridity and mechanical damages. The major HSPs synthesized by eukaryotes, including plants, belong to six conserved classes:HSP100, HSP90, HSP70, HSP60 and small (sm) HSPs. Among these, the most abundant in higher plants is a group of smHSPs. smHSPs with a molecular mass of 15 to 42 kDa, have conserved carboxyl-terminal heat-shock domain which is homologous to a-crystallins of the vertebrate eye lens. Plant sHsps are all encoded by nuclear genes and are divided into six classes. Two classes (classes CI and CII) of sHsps are localized in the cytosol and the other four in the chlorplastid(CP),the endoplasmic reticulum(ER), the mitochondria(Mt) and the peroxisomes(Po). Some sHsps have been demonstrated to act as molecular chaperone in vitro and in vivo,that is the ability to recognize and bind unfolded proteins, to suppress protein aggregation, and to influence the yield of protein folding are taken into account to define molecular chaperone activities.Based on the cDNA library constructed from Chimonanthus praecox flowers and EST analysis, a new member of heat shock proteins(HSPs) was cloned by randomly cloning and sequencing, named as CpHSP1 (GenBank accession No. HQ894379), and the bioinformatics, prokaryotic expression and real-time quantitative PCR were used to analyze its potential biological functions. The main results are as follows:1. Molecular characteristics of CpHSP1 The full length of CpHSP1cDNA sequence was 790 bp, with an open reading frame (ORF) of 477 bp encoding a putative polypeptide of 158 amino acid residues. Sequence alignments and phylogenetic analysis revealed CpHSP1 to be a cytosolic class I smHSP(small HSPs), which contained consensus I and II of ACD (a-crystallin domain) in the carboxyl-terminal (C-terminal) domain,and the conserved region specific for cytosolic class I smHSPs in the amino-terminal (N-terminal) domain.2. Prokaryotic expression of CpHSPlAfter being inserted into an prokaryotic expression vector pET32a(+),CpHSP1 was transformed into and overexpressed in E.coli BL21(DE3). The result of SDS-PAGE was indicated that the molecular weight of recombinant protein by IPTG induced was 37KDa wich coincided with our expectation.The growth curve of transformed and wild type cells when they were cultured at 37℃,but when the temperature being transferred to 50℃, which was known to cause cell autolysis, those cells with accumulated CpHSP1 exhibited improved viability compared with control. Similar results were observed under chilling treatment at 4℃as well. Overall, the results indicate that CpHSPl had an important influenee on cell viability of E.coliand responses to heat and chilling stress of plants.3. Transcriptional Expression of CpHSP1According to expression studies by real-time quantitative PCR, CpHSP1 was constitutively expressed in vegetative and reproductive organs at defined developmental stages under normal conditions, which was highly expressed in stems than other organs. CpHSP1 transcripts were detected during flower opening and more abundant in petals that in stamens and pistils. whereas CpHSP1 was hardly expressed in non-stressed leaf tissue but strongly induced under the the pressure of various abiotic stresses such as high temperature, low temperature, salinity, ABA and heavy metel. All these transcriptional expression analysis results suggested that CpHSP1 may play a significant role in plant development and abiotic stress responses.