Cloning and expression analysis of <italic>HSC</italic>70 gene in <italic>Trachidermus fasciatus</italic>

Abstract

Among heat shock proteins, heat shock protein 70(HSP70) is highly conserved in biological evolution, widely found in archaea, plants and animals, and plays an important role in biological resistance to external stimuli, folding and packaging of proteins, presenting antigens and cell proliferation and differentiation. It can be divided into two categories. One type can be induced to express rapidly and in large quantities under different stress conditions, but will be restored to the basic level under normal environment, so they are usually called induced HSP70. The other type has no stress induction process and is expressed constitutively, which is called heat shock homologous protein 70 (HSC70).In this study, a heat shock homologous protein (TfHsc70) was cloned from <italic>Trachidermus fasciatus</italic> by expressing sequence tag (EST) and rapid cDNA end amplification (RACE) technique. It was 2 138 bp with a theoretical molecular weight of 71.1 kDa and an isoelectric point of 5.27,and included an open reading frame (ORF) of 1 947 bp, a 5′-terminal untranslated region (UTR) of 78 bp, and a 3′-terminal UTR of 95 bp with a stop codon (TAA). Sequence alignment showed that <italic>TfHsc</italic>70 contained three conserved sequence tags of the HSP70 family (GIDLGTTY, DLGGGTFD and VLVGGSTRIPKIQK) and an ATP-GTP binding site motif (AEAYLGKT).Compared with the heat shock homologous protein 70 (HSC70) of many fishes, amphibians and reptiles, it was found that the similarity of HSC70 was 88.89%-97.09%. Quantitative real-time PCR (qRT-PCR) demonstrated that the <italic>TfHsc</italic>70 mRNA was expressed in blood, heart, liver, stomach, intestine, spleen, kidney, brain, and gill of <italic>Trachidermus fasciatus</italic>, and the expression level of the <italic>TfHsc</italic>70 mRNA was the highest in the brain. But it was almost undetectable in the skin. When using lipopolysaccharide (LPS) to stimulate <italic>Trachidermus fasciatus</italic>, the expression of <italic>TfHsc</italic>70 significantly increased in skin, blood and liver at 2 h, and was slightly upregulated in gill and spleen, but <italic>TfHsc</italic>70 showed initial down-regulation followed by a gradual up-regulation in the brain. This was due to the sensitivity of different tissues to LPS. Based on the analysis of the protein sequence of TfHsc70 and the expression of <italic>TfHsc</italic>70 mRNA, it was shown that TfHsc70 was a member of HSP70 family of heat shock proteins, and it might play an important role in innate immunity of <italic>Trachidermus fasciatus</italic>. In conclusion, the research explored the sequence characteristics and expression of <italic>TfHsc</italic>70 mRNA in <italic>Trachidermus fasciatus</italic>, which laid a foundation for the study of innate immunity in <italic>Trachidermus fasciatus</italic> and other fish.