Heat shock cognate 70 gene in Haliotis diversicolor: responses to pathogen infection and environmental stresses and its transcriptional regulation analysis

Yuting Li, Tao Zhang, Xin Zhang, Guodong Wang, Yilei Wang, Ziping Zhang

Heat shock cognate 70 (HSC70) is a class of highly conserved proteins which functions as a molecular chaperon, participates in tolerance processes, and is involved in protein folding, degradation, targeting, translocation, and protein complex remodeling. In this study, the mRNA expression level of the Haliotis diversicolor HSC70 (HdHSC70) gene was detected by quantitative real-time PCR in different tissues and under different stresses. The results showed that the HdHSC70 gene was ubiquitously expressed in seven selected tissues. The highest expression level was detected in gills (P < 0.05). The expression level of the HdHSC70 gene was significantly upregulated by thermal stress, hypoxia stress, Vibrio parahaemolyticus infection, and combined thermal and hypoxia stress. The upregulation occurred at the early stage of stress. These results indicated that the HdHSC70 is an important component in the immune system of H. diversicolor and is involved in the early stress response. Meanwhile, 5'-flanking region sequence (2013 bp) of the HdHSC70 gene was cloned; it contains a putative core promoter region, heat shock element, CpG, and transcription elements including NF-1, Sp1, Oct-1, interferon consensus sequence binding protein (ICSBP), etc. In HEK 293T cells, the 5'-flanking region sequence is able to drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. The promoter activity increased after high-temperature treatment, which may be the immediate reason why the expression of the HdHSC70 gene was significantly upregulated by thermal stress. After the ICSBP-binding site was mutated, we found the luciferase activity significantly reduced, which suggested that the ICSBP-binding site has a certain enhancement effect on the activity of the HdHSC70 promoter.