Li Jiuxuan, Zhang Haibin, Zhang Xiuyue, Yang Shiyong, Yan Taiming, Song Zhaobin
Sichuan Key Laboratory of Conservation Biology on Endangered Wildlife, College of Life Sciences, Sichuan University, Chengdu, 610065, People's Republic of China.
Fish Physiol Biochem. 2015 Apr;41(2):573-85. doi: 10.1007/s10695-015-0030-4. Epub 2015 Feb 18.
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.
通过逆转录聚合酶链反应（RT-PCR）和cDNA末端快速扩增技术，从齐口裂腹鱼（Schizothorax prenanti）肝脏中分离出两个编码热休克同源蛋白70（HSC70，命名为Sp-HSC70）和诱导型热休克蛋白70（HSP70，命名为Sp-HSP70）的互补脱氧核糖核酸（cDNA）克隆。这两个cDNA长度分别为2344 bp和2292 bp，包含1950 bp和1932 bp的开放阅读框，分别编码649个和643个氨基酸的蛋白质。氨基酸序列分析表明，Sp-HSC70和Sp-HSP70均包含HSP70家族的三个特征序列、两个部分重叠的双分型核定位信号序列（一个ATP结合位点基序、一个双分型核靶向信号）以及一个细胞质特征基序EEVD。同源性分析显示，Sp-HSC70与Sp-HSP70的同一性为77.5%，Sp-HSC70与其他脊椎动物已知HSC70的同一性超过81.1%，而Sp-HSP70与其他脊椎动物已知HSP70的同一性超过77.5%。荧光实时定量RT-PCR显示，在所有检测组织中均发现Sp-HSC70和Sp-HSP70 mRNA，包括血液、脑、心脏、肝脏、脾脏、头肾、白肌、皮肤、性腺、垂体、红肌和鳃。与对照组相比，感染嗜水气单胞菌24小时后，受弧菌攻击组血液和头肾中的Sp-HSC70和Sp-HSP70 mRNA表达水平显著升高。在时间上，细菌攻击后Sp-HSC70或Sp-HSP70基因存在明显的时间依赖性表达模式，Sp-HSC70和Sp-HSP70 mRNA的表达分别在攻击后12小时和6小时达到最高水平。7×24小时后两者均恢复到对照水平。结果表明，Sp-HSC70和Sp-HSP70基因可能在介导齐口裂腹鱼嗜水气单胞菌相关疾病的免疫反应中发挥重要作用。
