科研之友
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摘要
Heat shock proteins (HSPs) play crucial roles in protecting cells against environmental stresses, such as heat shock, heavy metals and pathogenic bacteria. Among the HSP family, the 70-kDa HSPs are most responsible for intracellular chaperone and extracellular immunoregulatory functions. The full-length HSP70 cDNA of Mytilus coruscus (designated as McHSP70, GenBank accession no. KF322135) was obtained, which was 2319 bp, including an ORF of 1965 bp to encode a polypeptide of 655 amino acid with predicted pl/MW5.29/71.38 kDa, a 81 bp-5'-UTR and a 270 bp-3'-UTR. The BLAST analysis and phylogenetic relationship strongly suggested that this amino acid sequence was a member of HSP70 family and highly homologous with Mytilus galloprovincialis (95%). Multiple sequence alignment revealed that McHSP70 and other known HSP70 were highly conserved, especially in the regions of HSP70 family signatures, the bipartite nuclear targeting sequence, ATP/GTP-Binding site motif and 'EEVD' motif. The mRNA of McHSP70 in hemolymph was constitutively expressed in all treatments including Vibrio challenge, thermal stress, metals (copper and cadmium) and 180 CST fuel exposure based on SYBR Green quantitative RT-PCR analysis. The temporal expression of HSP70 mRNA in hemolymph was significantly affected by Vibrio alginolyncus and Vibrio harveyi challenge. The maximum level appeared at 24 h post-injection with 5.44-fold in V. alginolyticus and dropped back to the original level at 72 h post-injection. In V. harveyi infection the transcripts of McHSP70 was significantly induced to the highest at 2 h after post-injection with 13.52-fold, then decreased until 36 h appearing another expression peak with 10.29-fold, and returned gradually to-physiological level at the end of this experiment. In heat shock experiment the maximum expression appeared at 12 h with 15-fold higher than that of blank mussels. Time-dependent mRNA expression pattern of McHSP70 was found in exposure to copper, cadmium and 180 CST fuel, but the highest expression and time were different in various treatments (copper of 9.41-fold at day 15, cadmium of 10.82-fold at day 10, and 180 CST fuel of 5.94-fold at day 25), and then dropped progressively. All these results indicated that HSP70 in marine bivalve had a significant role in mediating the environmental stress and immune response.
