Molecular characterization of GRP94 and HSP90α from Trachinotus ovatus, Linnaeus 1758 and their expression responses to various levels of stocking density stress and Cryptocaryon irritans infection

Abstract

Heat shock proteins (HSPs) are a class of molecular chaperones with a wide range of biological activities. To understand the stress responses of 94-kDa glucose-regulated protein (GRP94) and heat shock protein 90 alpha (HSP90α) under stocking density stress and C. irritans infection, we obtained their cDNA sequences from T. ovatus and performed bioinformatic analysis on their structures. Meanwhile, the tissue-specific expression patterns of these genes were also detected by qPCR. The ToGRP94 cDNA contains an open reading frame of 2394 bp, which encodes a protein of 797 amino acids, while ToHSP90α contains an open reading frame of 2190 bp, which encodes a protein of 730 amino acids. Amino acid sequence analysis showed that these proteins have highly conserved characteristic family domains or motifs, such as the heat shock hsp90 protein family signature (Y [k/s] NKEIFLRE). ToGRP94 was recently related to S. dumerili GRP94 with amino acid sequence homology up to 96.86%, and ToHSP90α had a higher evolutionary correlation with M. armatus and A. testudineus HSP90α with sequence homology of 98.49% and 97.67%, respectively. ToGRP94 and ToHSP90α mRNAs were widely expressed in all tissues tested under normal conditions. ToGRP94 mRNA was most strongly expressed in gill tissue followed by fin and kidney tissues, while ToHSP90α was most strongly expressed in blood tissues followed by brain, kidney and gill tissues. Under stocking density stress, ToGRP94 mRNA in the liver was significantly higher in the LD group, but it did not change in the kidney and spleen tissues with increasing stocking density; however, the expression level of ToHSP90α gene increased significantly in liver, spleen, and kidney tissues under high stocking densities, which indicates that ToHSP90α was closely related to density chronic stress. After C. irritans infection, the expression of ToGRP94 was significantly increased in liver, spleen, and kidney tissues. Similarly, ToHSP90α gene expression was significantly increased in liver and kidney tissues, but there was no difference in spleen tissue. The results indicate that these two genes may be involved in the immune response of T. ovatus. In summary, ToHSP90α would be involved in chronic stress and the immune response, while ToGRP94 would be mainly involved in the immune regulatory response. These results may provide a reference for further research studying the function of fish HSP90 and its mechanism under environmental stress.