Molecular cloning, expression HSP70 and its response to bacterial challenge and heat stress in Microptenus salmoides
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Molecular cloning, expression HSP70 and its response to bacterial challenge and heat stress in Microptenus salmoides Chunnuan Zhang & Kangle Lu & Junhui Wang & Qi qian & Xiaoyu Yuan & Changchang Pu
Received: 17 February 2020 / Accepted: 27 September 2020 # Springer Nature B.V. 2020
Abstract The gene encoding HSP70 was isolated from Microptenus salmoides by homologous cloning and rapid amplification of cDNA ends (RACE). The HSP70 transcripts were 2116 bp long and contained 1953 open reading frames encoding proteins of 650 amino acids with a molecular mass of 71.2 kDa and theoretical isoelectric point of 5.22. The qRT-PCR analysis showed that the HSP70 gene was differentially expressed in various tissues under normal conditions, and the highest HSP70 level was observed in the spleen and the lowest levels in the muscle and heart. The clear time-dependent expression level of HSP70 was observed after bacterial challenge and heat stress. A significant increase in HSP70 expression level was detected and reached a maximum at 3 h and 6 h in liver, spleens and gill tissues after Aeromonas hydrophila infection and heat stress, respectively (P < 0.05). As time progressed, the expression of HSP70 transcript was downregulated and mostly dropped back to the original level at 48 h. The concentration of cortisol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased as the time of stress progressed, with the highest level found on 3 h and later C. Zhang (*) : J. Wang : Q. qian : X. Yuan : C. Pu College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, People’s Republic of China e-mail: [email protected] K. Lu Laboratory of Aquatic Animal Nutrition and Physiology, Fisheries College, Jimei University, Xiamen 361021, China
declined rapidly and reached to the control levels at the 48 h. Those results suggested that HSP70 was involved in the immune response to bacterial challenge and heat stress. The cloning and expression analysis of the HSP70 provide theoretical basis to further study the mechanism of anti-adverseness in Microptenus salmoides. Keywords Microptenus salmoides . HSP70 gene . Immune . A. hydrophila challenge . Heat stress
Heat shock proteins (HSPs) consist of several families of highly conserved proteins found in all living organisms from bacteria to humans (Han et al. 2016). Based on HSPs’ apparent molecular weight, mass-amino acid sequence homologies and functions, HSPs are divided into five major subfamilies: HSP100, HSP90, HSP70, HSP60 and the small HSP family (Anne-Laure et al. 2009). Among the HSP families, HSP70 is an important member of the heat shock protein superfamily and appears in almost all species (Krenek et al. 2013). HSP70s are evolutionarily conserved proteins in all living organisms that play vital roles in environmental adaptation (Vergaraamado et al. 2017; Cheng et al. 2019). In addition to this, HSP70 proteins are also involved in improved resistance to disease. HSPs were extensively studied earlier and purified in most of
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