Cloning and expression analysis of two small heat shock proteins genes from Aquilaria sinensis / 中草药

Objective: To aim at cloning the open reading frame (ORF) of sHSP1 and sHSP2 genes from Aquilaria sinensis and analyzing the bioinformatics and expression of the two genes. Methods: Two unique sequences containing sHSPs domain were discovered in transcriptome dataset of A. sisnensis. The full-length cDNAs of sHSP1 and sHSP2 were cloned by RT-PCR strategy with the specific primers. Subcellular localization, transmembrane domain, three-dimensional structure, and phylogenetic analysis were predicted by different softwares to analyze the bioinformatics of sHSPs protein. The expression different levels of sHSP1 and sHSP2 isoforms in different tissues and in responds to salt and ABA, SA, MJ treatment were measured by real-time quantitative PCR. Results: The sHSP1 and sHSP2 cDNA sequence consisted of 474 bp ORF, encoding 157 amino acids. Tissue expression analysis indicated that sHSP1 and sHSP2 were primarily expressed in roots, followed by stems and leaves. Salt treatment experiments indicated that salt treatment caused a rapid increase in sHSP1and sHSP2 expression within 36 and 24 h, respectively. Exogenous ABA and MJ treatment experiments indicated that sHSP1 and sHSP2 genes were induced by exogenous ABA and MJ, and all reached the highest expression level at 12 h. Simultaneously, the SA treatment experiments indicated that exogenous SA treatment caused a rapid increase in sHSP1 and sHSP2 expression within 12 and 24 h, respectively. Conclusion: The full-length cDNA sequence of sHSP1 and sHSP2 genes from A. sinensis is obtained. sHSP1 and sHSP2 have the different expression level in different tissues. When subjected to high salt, ABA, SA, and MJ treatment, sHSP1 and sHSP2 show the different expression levels in different time. Cloning and analyzing sHSP1 and sHSP2 genes from A. sinensis will play an important role for further study on plant defense response.

Screening Molecular Chaperones Similar to Small Heat Shock Proteins in Schizosaccharomyces pombe

To screen molecular chaperones similar to small heat shock proteins (sHsps), but without alpha-crystalline domain, heat-stable proteins from Schizosaccharomyces pombe were analyzed by 2-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Sixteen proteins were identified, and four recombinant proteins, including cofilin, NTF2, pyridoxin biosynthesis protein (Snz1) and Wos2 that has an alpha-crystalline domain, were purified. Among these proteins, only Snz1 showed the anti-aggregation activity against thermal denaturation of citrate synthase. However, pre-heating of NTF2 and Wos2 at 70degrees C for 30 min, efficiently prevented thermal aggregation of citrate synthase. These results indicate that Snz1 and NTF2 possess molecular chaperone activity similar to sHsps, even though there is no alpha-crystalline domain in their sequences.

HSP12.1, A Small Heat Shock Protein in C.elegans, Has Chaperone-like Activity / 生物化学与生物物理进展

Many kinds of small heat shock proteins (sHSPs) are able to prevent protein aggregation in stress, which show the ATP independent chaperone-like activity. The smallest protein HSP12.1 in sHSP family of the nematode Caenorhabditis elegans exhibits chaperone-like activities in vitro. It prevents protein aggregation in a certain extent when use insulin, ADH and lysozyme as the substrates, though it is not as efficient as the typical chaperones (such as HSP16.1 in C. elegans). By contrast, the other three sHSP12s (HSP12.2, HSP12.3 and HSP12.6), which have similar molecular masses and primary structure, appear devoid of in vitro chaperone-like activities. In addition, overexpressing HSP12.1 enhances cell thermotolerance of Escherichia coli. The survival rate of the HSP12.1 overexpressed cells is 4-fold higher than the control, yet whether it does the same function in C. elegans is still unknown. Results indicate that C-terminal region is not necessary for the chaperone-like activity of sHSPs, for HSP12.1 terminates a short C-terminal tail. N-terminal domain may play a relatively important role in the exhibition of chaperone-like activities, while ?-crystalline domain may also involve in this function.