Molecular cloning and expression dynamics of UNC-45B upon heat shock in the muscle of yellowtail

Abstract

As a myosin chaperone, uncoordinated-45B (UNC-45B) is expressed specifically in striated muscle tissues. It has been proved that overheat shock will lead to myosin denaturation, and that UNC-45B is involved in myosin assembly. Yellowtail was chosen for this study, as an important economic aquaculture fish worldwide. To establish the heat shock response of UNC-45B in yellowtail, cDNA cloning and expression analysis were carried out. The cDNA of yellowtail unc-45b was successfully cloned, and consisted of 3051 bp encoding 930 amino acid residues (ORF 2793 bp). Based on the sequence of yellowtail unc-45b, we designed original primers and an anti-peptide antibody for our study. Yellowtail were then exposed to short-term or long-term heat shock, and UNC-45B expression levels were determined by qPCR and western blot analysis. For short-term heat shock exposure, the fish were kept in water tanks at 25 °C (control group) and 30 °C (heat shock group) for up to 30 h. The mRNA and protein expression levels of UNC-45B increased markedly after 8 h and 24 h, respectively. To investigate the effects of long-term heat shock exposure, cultured yellowtail were sampled in summer and winter at sea surface temperatures of 30 °C and 14 °C. The expression of UNC-45B in both mRNA and protein levels was significantly higher in summer than in winter. Our results show that the increase in UNC-45B expression levels can reflect heat shock response sensitively, and it is suggested that UNC-45B can repair the denatured myosin during heat shock exposure in yellowtail. Moreover, we successfully detected the protein expression of UNC-45B in aquaculture fish species in native conditions by using western blot analysis for the first time. Therefore, we assume that UNC-45B can be regarded as a biomarker to predict heat shock resistance level. This would help in aquaculture production, in the selection of juveniles able to endure heat shock.