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海带hsp70基因的克隆、分析及转录水平定量研究
袁艳敏1,2, 刘福利2,3, 梁洲瑞2, 汪文俊2, 孙修涛2, 王飞久2
1.上海海洋大学水产与生命学院 上海 201306;2.农业农村部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;3.青岛海洋科学与技术试点国家实验室 海洋渔业科学与食物产出过程功能实验室 青岛 266071
摘要:
本研究以海带(Saccharina japonica)‘海天1号’为材料,通过RACE-PCR方法获得海带hsp70(Sjhsp70)基因全长cDNA序列。序列分析表明,全长cDNA长度为3778 bp,含有1个2892 bp的开放阅读框,5ʹ和3ʹ非编码区的长度分别为101、785 bp。蛋白质氨基酸含量和理化性质分析显示,该蛋白为稳定蛋白。应用BLASTP程序对不同物种HSP70蛋白比较分析表明,海带与长囊水云(Ectocarpus siliculosus)的HSP70蛋白具有较高的同源性,用HSP70蛋白构建的进化树显示,海带与长囊水云聚为一类。二级结构预测该蛋白由963个氨基酸组成,包含35个α螺旋和25个β折叠。以酿酒酵母(Saccharomyces cerevisiae)HSP110蛋白三维结构3C7N为模板,在I-TASSER软件上预测海带HSP70蛋白的三维结构,预测的蛋白结构与3C7N的A链在折叠和二级结构方面非常类似。对Sjhsp70基因在温度胁迫下表达变化分析表明,高温对Sjhsp70的表达量具有显著影响,Sjhsp70在25℃、24 h胁迫条件时,表达量达到最大值。本研究为阐明海带HSP70蛋白特性及其应对环境胁迫的机理提供理论依据。
关键词:  海带‘海天1号’  HSP70  RACE  生物信息学分析  荧光定量PCR
DOI:
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基金项目:
Cloning, bioinformatics and quantitative expression analysis of hsp70 in Saccharina japonica
YUAN Yanmin1,2, LIU Fuli2,3, LIANG Zhourui2, WANG Wenjun2, SUN Xiutao2, WANG Feijiu2
1.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306;2.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;3.Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266071
Abstract:
HSP70s are heat shock proteins that exist widely in prokaryotes and eukaryotes, and play important roles in biological resistance. The full cDNA sequence of a hsp70 gene in Saccharina japonica (Sjhsp70) of 3778 bp was obtained by RACE technology. Sjhsp70 consisted of an open reading frame (ORF) of 2892 bp, a 5ʹ untranslated region (UTR) of 101 bp, and a 3ʹ UTR of 785 bp. Analysis of amino acid content and physicochemical properties showed that SjHSP70 is a stable protein. Comparison between the amino acid sequences of SjHSP70 and the well-characterized orthologous HSP70 proteins from various species indicated that SjHSP70 shares the highest similarity with the orthologous protein from Ectocarpus siliculosus. The phylogenetic tree of the examined proteins indicated that SjHSP70 formed a group with a HSP70 from E. siliculosus. Secondary structure analysis of SjHSP70 showed that it is composed of 35 α-helices and 25 β-strands. Three-dimensional homology structure modeling of SjHSP70 was predicted by the I-TASSER server according to the 3C7N template. Overall, the folding and secondary structures of SjHSP70 were highly similar to those of 3C7N. To study the correlation between SjHSP70 and the resistance to high-temperature stress in S. japonica, real-time variation in the expression of Sjhsp70 under different temperatures and different stress times was determined. The results showed that high temperature had a significant effect on the expression of Sjhsp70. The expression of Sjhsp70 continued to increase with increase in temperature, with the maximum Sjhsp70 expression at 25℃ after 24 h of high-temperature stress. This result indicates that Sjhsp70 is probably related to high-temperature resistance in S. japonica. This study provides a foundation for the characterization of SjHSP70 and for the study of SjHSP70 function in the high-temperature stress response in S. japonica.
Key words:  Saccharina japonica ‘Haitian No.1’  HSP70  RACE  Bioinformatics analysis  Quantitative real-time PCR