CLONING AND EXPRESSION ANALYSIS OF A HSC70 GENE FROM TURBOT

Disruption of normal cellular processes can cause the rapid and increased synthesis of a group of proteins belonging to the heat shock proteins (HSPs). Of all the HSPs, HSP70 has been widely studied as a biomarker of stress, and the major inducing factor for HSP70 up-regulation is the occurrence of damaged cellular protein. HSC70 (heat-shock cognate protein 70) is a constitutively expressed member of the 70 kD class of HSP70, which plays key roles in the cell as molecular chaperone and involves in a number of cellular processes. HSC70 has been shown to be involved in protein folding in the cytoplasm, protein import into the endoplasmic reticulum, mitochondria, chloroplasts, or trafficking of the receptors and coated vesicles and so on. In previous study, suppression subtractive hybridization (SSH) was used to investigate the response of turbot (Scophthalmus maximus) to Vibrio harveyi, using a cDNA library constructed from kidney and spleen of artificially infected turbot, and several immune-related genes were identified, including a hsp70 gene. In the present study, the complete cDNA sequence of turbot HSC70 was obtained using the method of RACE. The full length hsc70 cDNA of 2188 bp contained a 138 bp 5′-untranslated region (5′-UTR), a 1956 bp open reading frame (ORF) encoding 651 amino acids, and a 94 bp 3′-untranslated region (3′-UTR). The specific motif of Dnak (DLGTT-S-V, 10-18 aa), EEVD (648-651 aa) and GGMP repeated tetra-peptide (615-630 aa) were found in the deduced amino acid sequence of turbot HSC70. Compared with the members of HSC70 from other organisms, turbot HSC70 had 97%, 95%, 94%, 93% and 86% identity with HSC70 from flounder (Paralichthys olivaceus), rainbow trout (Oncorhynchus mykiss), human (Homo sapiens), mouse (Mus musculus) and tobacco (Manduca sexta) respectively. The overall topology of the phylogenetic tree showed that the turbot HSC70 and HSC70s from other fish formed one cluster. Quantitative real-time PCR demonstrated that hsc70 mRNA expressed constitutively in all of the test tissues, and the lowest expression level of turbot hsc70 mRNA was detected in muscle, the highest expression level was in liver by 45.4-fold. After the injection of physiological saline, the hsc70 mRNA expression levels in liver and spleen had no significant changes compared to those of non-injected turbot (P>0.05). This suggested that the injection itself had no influence on the expression of hsc70. After turbot were challenged with V. harveyi, the expression levels of hsc70 mRNA were up-regulated in liver (2.5-fold) and spleen (1.6-fold) at 24h and 12h, respectively (P<0.05). The results provided a preliminary foundation for the further functional research of turbot HSC70.