热胁迫下鹿角杯形珊蝴HSP70的基因表达与活性分析
发布时间：2018-07-21 17:20
【摘要】：珊瑚礁生态系统是海洋生态系统的重要组成部分。当前,全球海水升温严重威胁着珊瑚礁生态系统,造成了大量珊瑚白化死亡。造礁石珊瑚鹿角杯形珊瑚Pocillopora damicornis不仅被列为全球气候变化模拟研究中的生物模型,还被认为是珊瑚生态适应研究中的模式生物。因此,探索鹿角杯形珊瑚的热胁迫分子机制具有非常重要的意义。热激蛋白HSP70参与生物体对热胁迫的抵抗,能辅助机体恢复异常蛋白的正常构象并维持细胞的稳态。本研究以鹿角杯形珊瑚HSP70(PdHSP70)为研究对象,探索PdHSP70在热胁迫下的表达响应及其生物学活性特征。本研究利用RACE技术克隆获得了 PdHSP70 cDNA的全长序列。PdHSP70基因全长 2346 bp,其中 5' UTR(Untranslated region)长为 103 bp,3' UTR 为 290 bp,ORF(Open reading frame)为1953 bp,推导的多肽含650个氨基酸,分子量为71.93 kDa。利用SMART技术预测PdHSP70蛋白含有一个HSP70结构域(8-616氨基酸区域)。利用InterPro方法预测PdHSP70有三个结构域:ATPase结构域(actin-like ATPase domain,8-189 和 192-385 位氨基酸区域)、多肽结合结构域(peptide-binding domain,390-547位氨基酸区域)和C末端结构域(C-terminal domain,523-623位氨基酸区域)。利用荧光实时定量RT-PCR技术,检测了热胁迫(32℃)后PdHSP70 mRNA的表达变化。高温胁迫后,PdHSP70mRNA的表达水平在12小时显著提高,在24小时降回至正常水平。对照组中PdHSP70 mRNA的表达水平在24小时内没有显著变化。构建重组表达载体后,在大肠杆菌中诱导表达了具有ATPase活性的PdHSP70重组蛋白。在25℃、30℃、35℃和40℃条件下,PdHSP70重组蛋白的ATPase活性分别为21.76、21.88、13.60和16.20 nmol min-1 mg-1,且未发现显著差异。本研究表明,鹿角杯形珊瑚在热胁迫条件下会大量表达PdHSP70,来清除细胞中产生的错误折叠蛋白,并维持细胞的正常生理功能。当热胁迫撤除或热适应后,PdHSP70将恢复正常表达水平,以恢复机体的内稳态。PdHSP70作为一种热诱导型蛋白,在鹿角杯形珊瑚的热适应和内稳态维持中发挥着非常重要的作用。PdHSP70的相关研究初步揭示了造礁石珊瑚的热胁迫分子机制,为珊瑚礁生态系统的保护提供了理论基础,并丰富和发展了热带海洋生物学的相关内容。
[Abstract]:Coral reef ecosystem is an important part of marine ecosystem. At present, the global warming of sea water seriously threatens coral reef ecosystem, resulting in a large number of coral bleaching death. Coral corals Pocillopora damicornis is not only listed as a biological model in global climate change simulation, but also considered as a model organism in the study of coral ecological adaptation. Therefore, it is of great significance to explore the molecular mechanism of heat stress in corals. Heat shock protein (HSP70) is involved in the resistance of organism to heat stress, which can assist the body to restore the normal conformation of abnormal protein and maintain the homeostasis of cells. In this study, the expression response and biological activity of PdHSP70 under heat stress were studied. In this study, the full-length sequence of PdHSP70 cDNA. PdHSP70 gene was cloned by race technique. The total length of PdHSP70 gene was 2346 BP, in which the length of 5'UTR (untranslated region) was 103bpf3'UTR was 290bp ORF (Open reading frame) = 1953 BP). The deduced peptide contained 650 amino acids and its molecular weight was 71.93kDa. Smart technique was used to predict that the PdHSP70 protein contained a HSP70 domain (8-616 amino acid region). The three domains of PdHSP70 (actin-like domain 8-189 and 192-385 amino acid domain), polypeptide binding domain (peptide-binding domain 390-547 amino acid domain) and C-terminal domain (C-terminal domain 523-623 amino acid domain) were predicted by InterPro method. The changes of PdHSP70 mRNA expression under heat stress (32 鈩，
本文编号：2136238
[Abstract]:Coral reef ecosystem is an important part of marine ecosystem. At present, the global warming of sea water seriously threatens coral reef ecosystem, resulting in a large number of coral bleaching death. Coral corals Pocillopora damicornis is not only listed as a biological model in global climate change simulation, but also considered as a model organism in the study of coral ecological adaptation. Therefore, it is of great significance to explore the molecular mechanism of heat stress in corals. Heat shock protein (HSP70) is involved in the resistance of organism to heat stress, which can assist the body to restore the normal conformation of abnormal protein and maintain the homeostasis of cells. In this study, the expression response and biological activity of PdHSP70 under heat stress were studied. In this study, the full-length sequence of PdHSP70 cDNA. PdHSP70 gene was cloned by race technique. The total length of PdHSP70 gene was 2346 BP, in which the length of 5'UTR (untranslated region) was 103bpf3'UTR was 290bp ORF (Open reading frame) = 1953 BP). The deduced peptide contained 650 amino acids and its molecular weight was 71.93kDa. Smart technique was used to predict that the PdHSP70 protein contained a HSP70 domain (8-616 amino acid region). The three domains of PdHSP70 (actin-like domain 8-189 and 192-385 amino acid domain), polypeptide binding domain (peptide-binding domain 390-547 amino acid domain) and C-terminal domain (C-terminal domain 523-623 amino acid domain) were predicted by InterPro method. The changes of PdHSP70 mRNA expression under heat stress (32 鈩，
本文编号：2136238
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