Cloning, Expression And Functional Characterizations Of Small Heat Shock Protein Genes From Hevea Brasiliensis

The heat shock Proteins (HSPs) are highly conserved proteins synthesized by organisms stimulated by physical, chemical and biological stresses. According to their approximate molecular mass, HSPs are divided into five groups:HSP100family, HSP90family, HSP70family, HSP60family and the small heat shock protein (sHSP). sHSPs almost exist in all organisms. Acting as molecular chaperones, sHSPs can be coupled with partially folded or denatured substrate proteins to avoid them irreversible molecular aggregation or misfolding. In recent years, sHSPs have become one of the most important research areas within the field of plant stress resistance. In this study, two sHSPs genes, named as HbsHSP14and HbsHSPl7, were cloned from rubber tree. The bioinformatics characterizations, expression and functions of HbsHSP14and HbsHSP17were further analyzed. The main results are as follows:According to the known sequences of HbsHSP14and HbsHSP17obtained from assembled transcriptome data of rubber tree, the primers of5’and3’RACE were designed to amplify the sequences of the3’-and5’-end sequences of HbsHSP14and HbsHSP17cDNA with RACE method. After aligning and assembling the3’-and5’-end sequences and the known EST sequences, we obtained and confirmed the full-length cDNA sequence of HbsHSP by sequencing their PCR products. The full-length cDNA of HbsHSP14was991bp in size, and5’-and3’-UTR were206bp and30bp, respectively. The longest ORF of HbsHSPl4encodes a polypeptide of208amino acids with a calculated molecular mass of23.82kDa. The full-length cDNA of HbsHSP17was1002bp in size, and5’-and3’-UTR were separately202bp and155bp. The longest ORF of HbsHSP17encodes a polypeptide of214amino acids with a calculated molecular mass of23.85kDa. Bioinformatics analyses show that HbsHSP14and HbsHSP17cotained conserved alpha-crystallin domains (ACD). Within ACD,11and10polypeptide binding sites were separately predicted in HbsHSP14and HbsHSP17. These characterizations are very important for sHSPs to carry out their functions. HbsHSP14and HbsHSP17were separately predicted to be located in mitochondrion and chloroplast with TargetP analysis. Being different from the results of Target analysis, the phylogenetic analysis indicated that HbsHSP14and HbsHSP17were divided into sHSPs located into mitochondria (class M).The expressions of HbsHSP14and HbsHSP17showed significant differences in different tissues by real time RT-PCR analyses. HbsHSP14and HbsHSP17were both highly expressed in latex, followed by male flowers, female flowers, stem apex, leaves, barks. The expressions of HbsHSP14and HbsHSP17significantly varied depending on leaves development, the highest expression of HbsHSP14was light green young leaves; the expression levels in other four development stages were similar to each other; As for HbsHSP17, the highest and lowest expressions were separately in red and mature leaves. The expressions of HbsHSPl4and HbsHSP17were regulated by several stresses including wounding, H2O2, NaCl and low temperature. The statistical analyses showed that the expression changes of HbsHSP14and HbsHSP17indicated significant differences under all treatments except HbsHSP17under low temperature and two genes under wounding treatment. In addition, HbsHSP14and HbsHSP17were prominently regulated by ET and MeJA treatments, but the expression patterns of two genes were different. Under ET and MeJA treatments(expcet the time of treatments for72h), the expression of HbsHSP14firstly increased with the highest level in24h, and then decreased. HbsHSP17indicated fluctuant expression profiles with ET and MeJA treatments, with the lowest level in4h and the highest in48h. In contrast, the expressions of HbsHSP14and HbsHSP17did not significantly change in their corresponding control materials.The prokaryotic expression vector, pET28a(+)-HbsHSP14and pET28a (+)-HbsHSP17were successfully constructed and transformed into E. coli BL21(DE3). After inducing by1mM isopropyl-β-D-thiogalactopyranoside (IPTG), HbsHSP14/HbsHSP17and His tag recombinant proteins were expressed in E. coli BL21(DE3), and the molecular masses of recombinant proteins were consistent with their expected ones. By comparing the viability of E. coli overexpressing pET28a(+)-HbsHSP14and pET28a (+)-HbsHSP17and E. coli overexpressing pET28a under stresses, we found that HbsHSP14and HbsHSP17can enhance the viability of E. coli under high temperature and salt stresses. Under low temperature, H2O2, CuSO4, ZnSO4treatments, the viability of E. coli overexpressing pET28a(+)-HbsHSP14and pET28a (+)-HbsHSP17and E. coli overexpressing pET28a did not indicate significant difference.