【摘要】：珊瑚礁生態(tài)系統(tǒng)是海洋生態(tài)系統(tǒng)的重要組成部分。當前,全球海水升溫嚴重威脅著珊瑚礁生態(tài)系統(tǒng),造成了大量珊瑚白化死亡。造礁石珊瑚鹿角杯形珊瑚Pocillopora damicornis不僅被列為全球氣候變化模擬研究中的生物模型,還被認為是珊瑚生態(tài)適應(yīng)研究中的模式生物。因此,探索鹿角杯形珊瑚的熱脅迫分子機制具有非常重要的意義。熱激蛋白HSP70參與生物體對熱脅迫的抵抗,能輔助機體恢復(fù)異常蛋白的正常構(gòu)象并維持細胞的穩(wěn)態(tài)。本研究以鹿角杯形珊瑚HSP70(PdHSP70)為研究對象,探索PdHSP70在熱脅迫下的表達響應(yīng)及其生物學活性特征。本研究利用RACE技術(shù)克隆獲得了 PdHSP70 cDNA的全長序列。PdHSP70基因全長 2346 bp,其中 5' UTR(Untranslated region)長為 103 bp,3' UTR 為 290 bp,ORF(Open reading frame)為1953 bp,推導的多肽含650個氨基酸,分子量為71.93 kDa。利用SMART技術(shù)預(yù)測PdHSP70蛋白含有一個HSP70結(jié)構(gòu)域(8-616氨基酸區(qū)域)。利用InterPro方法預(yù)測PdHSP70有三個結(jié)構(gòu)域:ATPase結(jié)構(gòu)域(actin-like ATPase domain,8-189 和 192-385 位氨基酸區(qū)域)、多肽結(jié)合結(jié)構(gòu)域(peptide-binding domain,390-547位氨基酸區(qū)域)和C末端結(jié)構(gòu)域(C-terminal domain,523-623位氨基酸區(qū)域)。利用熒光實時定量RT-PCR技術(shù),檢測了熱脅迫(32℃)后PdHSP70 mRNA的表達變化。高溫脅迫后,PdHSP70mRNA的表達水平在12小時顯著提高,在24小時降回至正常水平。對照組中PdHSP70 mRNA的表達水平在24小時內(nèi)沒有顯著變化。構(gòu)建重組表達載體后,在大腸桿菌中誘導表達了具有ATPase活性的PdHSP70重組蛋白。在25℃、30℃、35℃和40℃條件下,PdHSP70重組蛋白的ATPase活性分別為21.76、21.88、13.60和16.20 nmol min-1 mg-1,且未發(fā)現(xiàn)顯著差異。本研究表明,鹿角杯形珊瑚在熱脅迫條件下會大量表達PdHSP70,來清除細胞中產(chǎn)生的錯誤折疊蛋白,并維持細胞的正常生理功能。當熱脅迫撤除或熱適應(yīng)后,PdHSP70將恢復(fù)正常表達水平,以恢復(fù)機體的內(nèi)穩(wěn)態(tài)。PdHSP70作為一種熱誘導型蛋白,在鹿角杯形珊瑚的熱適應(yīng)和內(nèi)穩(wěn)態(tài)維持中發(fā)揮著非常重要的作用。PdHSP70的相關(guān)研究初步揭示了造礁石珊瑚的熱脅迫分子機制,為珊瑚礁生態(tài)系統(tǒng)的保護提供了理論基礎(chǔ),并豐富和發(fā)展了熱帶海洋生物學的相關(guān)內(nèi)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，

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