Cloning And Expression Analysis Of Hsp70 Gene From Freshwater Planarian Dugesia Japonica

Heat shock protein 70 (HSP70) is an important member of the heat shock protein family. Many studies have confirmed that HSP70 play a key role in protein folding, membrane transport and the degradation of misfolded proteins, and is closely related to cell proliferation, cell differentiation and construction of the neural system. In addition, its expression was influenced by environmental stress and physiological stress. As an important protection protein, HSP70 has not been reported in planarians'adaptive stress response.In this study, the full-length hsp70 cDNA was firstly isolated and sequenced from planarian Dugesia japonica (Djhsp70) using RACE technology, Which was registered in GenBank with accession no.EU380241. The length of the Djhsp70 ORF from planarian genomic DNA and cDNA was identical, which indicated the absence of introns in the Djhsp70 gene.The full-length cDNA of Djhsp70 is 2066 bp containing an open reading frame (ORF) of 1947 bp encoding a polypeptide of 648 amino acids with a predicted molecular mass of 71.18 kDa. A sequence homology search using BLASTN and BLASTP reveals that DjHSP70 has high homology with other known HSP70s. The deduced amino acid sequence shares three signatures motifs(IDLGTTYS,DLGGGTFD,IVLVGG) and the C-terminal consensus EEVD of eukaryotic HSP70 family. This is consistent with the characteristics of HSP70 reported. According to the reported amino acid sequence of HSP70, a phylogenetic tree was constructed. The planarian is most closely related to M. galloprovincials (both belong to Lophotrochozoa), and has a closer relationship to vertebrates. But it was not clustered near C. elegans and D. melanogaster, the results displayed in the phylogenic tree were generally in agreement with the newly proposed taxonomy.Semi-quantitative RT-PCR was used to detected the expression levels of Djhsp70 mRNA in response to stressors. The results showed that its expression was upregulated in response to heat-stimulated, starvation-induced and Cutting injury. Planarians that are starvated for one week and fed on the 18℃were used as the normal control group. In the heat-stimulated condition, Djhsp70 expression in 10℃culture condition was slightly higher than in the control groups, but its expression levels were significantly increased (P﹤0.05) when elevating the culture temperature to 30℃for 24hours. When gradually elevating the culture temperatureto 32℃for 48hours, the expression was increased nearly 2-fold higher than control groups. In the starvation-induced condition, Djhsp70 expression had no significant difference from the control samples after 15 days starvation. After 30 days starvation, the expression increased gradually, and maintained relatively high levels during de-growth. Interestingly, Djhsp70 expression decreased drastically in the re-fed animals after 60 days starvation. In the cut injury condition, Djhsp70 expression increased apparently at 12 hours after the cut (P﹤0.05), and rose to the maximum at 36 hours. But it declined when the planarians were cut after 46 hours. We believe that DjHSP70 is inducible HSP and closely related to the planarians'heat resistance, fight hunger and regeneration of tissue injury.In order to further study the subcellular location of DjHSP70, expression vector encoding the C-terminal polypeptide of DiHSP70 was constructed, and expressed the fusion protein by the IPTG induced. The fusion protein molecular weight is about 21kDa which is consistent with expected. The fusion protein was purified by Ni-Agarose ,electrophoresis showed that the purity of the purified fusion protein is high, and the grayscale scanning revealed that the purity was over 95%. New Zealand rabbits were immunized with the purified fusion protein, and to raise polyclone antibody against HSP70. Western blotting found that the polyclone antibody can specifically combine to the HSP70 of planarian and have no cross reaction to other heat shock proteins. The titre of the antibody was detected by Dlot blot, and the result was 1:500. The antibody was used to detect the expression of DjHSP70 in response to heat-stimulated, starvation-induced and cutting injury, the results were basically consistent with the RT-PCR results.In conclusion, the full-length hsp70 cDNA was firstly cloned from the freshwater planarians Dugesia japonica and verified that it was a inducible hsp70 gene without the introns. It have been proved that Djhsp70 is closely related to the planarians'heat resistance, fight hunger and regeneration of tissue injury. The preparation of high-titer antibody laid a solid foundation for the further study on the subcellular location of HSP70 in planarians, and other biological functions and mechanism of action.