title: Molecular cloning and expression of two heat-shock protein genes (HSC70/HSP70) from Prenant's schizothoracin (Schizothorax prenanti).
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.
authors.affiliation: Sichuan Key Laboratory of Conservation Biology on Endangered Wildlife, College of Life Sciences, Sichuan University, Chengdu, 610065, People's Republic of China.
authors.forename: Jiuxuan
authors.initials: J
authors.lastname: Li
authors.forename: Haibin
authors.initials: H
authors.lastname: Zhang
authors.forename: Xiuyue
authors.initials: X
authors.lastname: Zhang
authors.forename: Shiyong
authors.initials: S
authors.lastname: Yang
authors.forename: Taiming
authors.initials: T
authors.lastname: Yan
authors.forename: Zhaobin
authors.initials: Z
authors.lastname: Song
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003530:Cyprinidae / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005453:Fluorescence; D020869:Gene Expression Profiling / Q000662:veterinary; D050883:HSC70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D019105:Random Amplified Polymorphic DNA Technique; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000662:veterinary
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins