Molecular characteristics of a novel HSP60 gene and its differential expression in Manila clams (Ruditapes philippinarum) under thermal and hypotonic stress.

The Manila clam Ruditapes philippinarum inhabits the intertidal zone and must therefore tolerate broad fluctuations in water temperature and salinity. Heat shock protein 60 (HSP60) is an evolutionarily conserved, multi-functional protein that plays a significant role in protecting organisms from harmful stress conditions. We cloned the R. philippinarum HSP60 (RpHSP60) gene and analyzed its transcriptional responses to thermal and low-salinity stresses. The complete sequence of RpHSP60 cDNA was 1777 nucleotides, containing a 1728-bp open reading frame encoding a polypeptide of 576-amino acids, with a calculated molecular mass of 61.25 kDa and predicted isoelectric point of 5.08. Comparisons of amino acid sequences and three-dimensional structures of HSP60 revealed that RpHSP60 was highly conserved in the signature HSP60-family domains. RpHSP60 mRNA was detected in all the tested tissues of R. philippinarum, with the highest expression levels in hemocytes. We measured RpHSP60 mRNA levels in the gills under thermal and low-salinity stresses using quantitative real-time reverse transcription-polymerase chain reaction. Following the thermal challenge, RpHSP60 mRNA was significantly upregulated at 6 h, and then progressively downregulated under high-temperature stress (30 °C), while only slight fluctuations were observed under low-temperature stress (-1 °C). Under low-salinity (17 ppt) stress, RpHSP60 mRNA levels were significantly increased at 3, 72, and 96 h (P < 0.05). These results suggest that HSP60 of R. philippinarum may play important roles in responding to high-temperature and low-salinity stresses.