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Molecular cloning and expression of two heat-shock protein genes (HSC70/HSP70) from Prenant’s schizothoracin (Schizothorax prenanti)

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Abstract

Through the RT-PCR and rapid amplification of cDNA ends, two complementary deoxyribonucleic acid (cDNA) clones encoding heat-shock cognate 70 (HSC70, designated Sp-HSC70) and inducible heat-shock protein 70 (HSP70, designated Sp-HSP70) were isolated from the liver of Prenant’s schizothoracin (Schizothorax prenanti). The cDNAs were 2344- and 2292-bp in length and contained 1950- and 1932-bp open reading frames, encoded proteins of 649 and 643 amino acids, respectively. Amino acid sequence analysis indicated that both Sp-HSC70 and Sp-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (an ATP-binding site motif, a bipartite nuclear targeting signal), and a cytoplasmic characteristic motif EEVD. Homology analysis revealed that Sp-HSC70 and Sp-HSP70 shared 77.5 % identity and Sp-HSC70 shared more than 81.1 % identity with the known HSC70s of other vertebrates, while Sp-HSP70 shared more than 77.5 % identity with the known HSP70s of other vertebrates. Fluorescent real-time quantitative RT-PCR showed that Sp-HSC70 and Sp-HSP70 mRNAs were found in all tested tissues, including blood, brain, heart, liver, spleen, head kidney, white muscle, skin, gonad, hypophysis, red muscle, and gill. The Sp-HSC70 and Sp-HSP70 mRNA expression level in blood and head kidney displayed a significant increase in vibrio-challenged group with the bacterium Aeromonas hydrophila at 24 h post-infection compared to a control group. Temporally, there was a clear time-dependent expression pattern of Sp-HSC70 or Sp-HSP70 gene after bacterial challenge, and the expression of Sp-HSC70 and Sp-HSP70 mRNAs reached a maximum level at 12 and 6 h post-challenge, respectively. Both returned to control level after 7 × 24 h. The results suggest that Sp-HSC70 and Sp-HSP70 genes may play important roles in mediating the immune responses of A. hydrophila-related diseases in the Prenant’s schizothoracin.

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Acknowledgments

This work was supported by the Program for New Century Excellent Talents in University (No. NCET-11-0347). We would like to thank Dr. Yi Geng for providing Aeromonas hydrophi1a and suggestion for the challenge experiment, and Dr. Peter S. Rand for his revision of English writing.

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    1. Sichuan Key Laboratory of Conservation Biology on Endangered Wildlife, College of Life Sciences, Sichuan University, Chengdu, 610065, People’s Republic of China

      Jiuxuan Li, Haibin Zhang, Xiuyue Zhang & Zhaobin Song

    2. Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, People’s Republic of China

      Xiuyue Zhang

    3. Department of Aquaculture, Sichuan Agricultural University, Ya’an, 625014, People’s Republic of China

      Shiyong Yang & Taiming Yan

    4. Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, 610065, People’s Republic of China

      Zhaobin Song

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    Correspondence to Zhaobin Song.

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    Jiuxuan Li and Haibin Zhang have contributed equally to this work.

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    Li, J., Zhang, H., Zhang, X. et al. Molecular cloning and expression of two heat-shock protein genes (HSC70/HSP70) from Prenant’s schizothoracin (Schizothorax prenanti). Fish Physiol Biochem 41, 573–585 (2015). https://doi.org/10.1007/s10695-015-0030-4

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