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张勇,徐钢春,杜富宽,顾若波.2016.美洲鲥hsp70的分子特征及其对运输应激的应答.动物学杂志,51(2):268-280.
美洲鲥hsp70的分子特征及其对运输应激的应答
Molecular Characterization of Hsp70 and Its Response to Transport Stress in American Shad (Alosa Sapidissima)
投稿时间:2015-09-09  修订日期:2016-02-24
DOI:DOI: 10.13859/j.cjz.201602013
中文关键词:  美洲鲥  热应激蛋白70  基因克隆  基因表达  运输应激
英文关键词:American shad (Alosa sapidissima)  Heat stress protein 70 (hsp70)  Gene cloning  mRNA expression  Transport stress
基金项目:国家科技支撑计划项目(No.2012BAD25B07); *通讯作者,E-mail:gurb@ffrc.cn;第一作者介绍 张勇,男,硕士研究生;研究方向鱼类繁育生理学;E-mail:846164414@qq.com。
作者单位E-mail
张勇 南京农业大学无锡渔业学院 young4321@163.com 
徐钢春 中国水产科学研究院淡水渔业研究中心农业部淡水渔业和种质资源利用重点实验室  
杜富宽 中国水产科学研究院淡水渔业研究中心农业部淡水渔业和种质资源利用重点实验室  
顾若波 中国水产科学研究院淡水渔业研究中心农业部淡水渔业和种质资源利用重点实验室  
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中文摘要:
      运用同源克隆和cDNA末端快速扩增(RACE)技术获取了美洲鲥(Alosa sapidissima)热应激蛋白70(hsp70)的全长cDNA序列,其总长度为2 545 bp,开放阅读框(ORF)为1 914 bp,推测编码637个氨基酸,其氨基酸的二级结构含有HSP70家族的3个特征序列。氨基酸同源性分析显示,美洲鲥hsp70与墨西哥脂鲤(Astyanax mexicanus)等鱼类的相似性达84%以上,与无脊椎动物果蝇(Drosophila auraria)及大肠杆菌(Escherichia coli)的相似性分别为73%和38%。荧光定量PCR检测显示,该基因在美洲鲥鳃、肌肉、头肾中高表达,在脑和心中相对高表达,在肝、脾、肠、肾中微量表达。采用荧光定量PCR方法研究运输应激对美洲鲥未经繁殖的亲鱼hsp70 mRNA水平的影响,在运输实验中鳃、肝hsp70 mRNA水平呈先升高后降低的变化趋势,头肾hsp70 mRNA水平呈现递增趋势。该结果表明克隆到的基因符合诱导型hsp70的特点,而且美洲鲥鳃、肝、头肾组织的hsp70 mRNA对运输应激表现出明显的应答作用。
英文摘要:
      American shad (Alosa sapidissima) belongs to Clupeomorpha, Clupeiformes, Clupeidae, Alosa. As eurythermal, euryhaline anadromous and migratory fish, American shad and Chinese shad are not only very similar in appearance and habits, but also have high nutritional value and economic value. American shad is extremely sensitive to changes in the external environment, vulnerable to the impact of environmental factors change (such as sound, light, water physical and chemical factors, etc.) and the influence of manual operation such as fishing, transportation, etc. Heat stress proteins (HSPs), also known as heat shock proteins, are founded that it is widely present in different species. It is a family of non-specific cell protective protein associated with resilience, and similar to a number of housekeeping genes. And the heat stress protein, especially the heat stress protein 70 (hsp70) family, is highly conserved in evolution. It also has a variety of biological functions: As a molecular chaperone, it assists proteins in properly folding, assembling, transporting, and regulating, repairing damaged proteins, and disintegrating of denatured proteins under stress; In cell protection, it can protect mitochondria from cytokines damage, play an important role in the anti-apoptotic, anti-oxidation and immune response. Besides, it also participates in the immune response in antigen-presenting, synergistic immune function. Hsp70 cDNA was cloned, analyzed, and the expression level of hsp70 mRNA was detected in A variety of fish species, such as bluntnose black bream (Megalobrama amblycephala), grass carp (Ctenopharyngodon idellus), silver carp (Hypophthalmichthys molitrix), nile tilapia (Tilapia nilotica), roughskin sculpin (Trachidermus fasciatus), bronze gudgeon (Coreius guichenoti ), siberian sturgeon (Acipenser baerii), sword tali (Xiphophorus hellerii), bastard halibut (Paralichthys olivaceus) etc. Studies show that the expression of hsp70 gene is regulated at the transcriptional level. The experiment, by the study on the expression of hsp70 gene in American shad broodstocks tissues and differential expression before and after the transport stress, aimed at a further understanding of the American shad in HSP70 protein synthesis and expression and regulation mechanism, in order to provide a theoretical basis for resistance mechanism study of American shad. In this experiment, by means of artificial transportation stress, we take 6 tails as the transport 0 h sampling, then catch 24 tails randomly divided into 12 bags, each bag 2 tails: 6 bags for 2 h transport stress sampling, while the other 6 bags for 4 h sampling. The differences between groups were analyzed by ANOVA One-way, and the results were expressed by Mean±SE. The full-length cDNA of hsp70 of American shad was cloned by reverse transcription poly merase chainreaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The results revealed that the length of hsp70 cDNA is 2 545 bp, containing an open reading frame (ORF) of 1 914 bp specified a peptide of 637 amino acids. The secondary structures contained three signature sequences of HSP70 family (Fig. 2). The homology analysis indicated that hsp70 of American shad shared more than 84.0% identity with the hsp70s of other fishes such as Astyanax mexicanus , it is also shared as high as 73% and 38% identity with Drosophila auraria and Escherichia coli (Tab. 2). The tissue-specific expressions of hsp70 mRNAs were detected in American shad. A high expression was observed in the gill, muscle and head kidney,while relatively high expression was encountered in brain and heart,and a weak expression in the liver, spleen, intestine and kidney (Fig. 4). Utilizing real-time quantitative PCR studied the effects of transport stress on expression of hsp70 mRNA in parent fish of American shad. Result of showed that hsp70 mRNA content in gill and liver increased significantly after transport 2 hours and then dropped after transport 4 hours (Fig. 5 and Fig. 6), in head kidney showing an increasing trend (Fig. 7). Results showed the gene is consistent with the characteristics of hsp70. And the hsp70 mRNA in gill, liver, head kidney showed obvious response on transport stress.
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