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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生命科學系所 > 依資料類型分類 > 碩博士論文 >  高鹽甲烷太古生物之熱休克蛋白DnaK、DnaJ和GrpE基因的選殖與特性分析

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152370

標題: 高鹽甲烷太古生物之熱休克蛋白DnaK、DnaJ和GrpE基因的選殖與特性分析
Cloning, Gene Expression, and Functional Characterization of Heat Shock Protein DnaK, DnaJ, and GrpE in Methanohalophilis portucalensis FDF1T
作者: 陳仕珊
Chen, Shih-Shan
Contributors: 賴美津
Mei-Chin Lai
生命科學系所
關鍵字: 太古生物;分子伴護蛋白;高鹽甲烷菌
Archaea;dnaJ;dnaK;grpE;Halophilic methanogen;Molecular chaperone
日期: 2012
Issue Date: 2013-11-07 13:23:21 (UTC+8)
Publisher: 生命科學系所
摘要: 細胞在面臨環境逆境時,胞內蛋白可能會失去正常功能和結構。分子伴護蛋白系統在生理上扮演修復失活蛋白的角色,DnaK/DnaJ/GrpE (KJE)需要依賴ATP幫助新生成蛋白摺疊或可與分子伴護因子GroEL/ES和AAA+家族的ClpB,將蛋白去聚集化再重新摺疊受逆境壓力而變性的蛋白。真核生物和細菌中廣泛的存在KJE,但是在太古生物中只在部分高溫和中溫菌發現,同時研究非常得少。高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T可生長在鹽濃為1.2到2.9 M的環境,利用自體生合成甜菜鹼 (glycine betaine) 做為滲透壓保護物質,也具有分子伴護蛋白基因clpB及groEL/S,基因表現量受到鹽度和溫度的影響。本研究以聚合酶連鎖反應和南方墨點法選殖高鹽甲烷太古生物M. portucalensis FDF1T 約3.6 kb大小的grpE-dnaK-dnaJ基因,並以反轉錄聚合酶連鎖反應和北方墨點法證實grpE-dnaK-dnaJ基因為共同轉錄單元體。由胺基酸序列分析MpDnaK在N端上具有EXXKXXXXS的ATP 鍵結位和C端受質蛋白鍵結位 (residues L366, V368, T369, L371, A387, L458),MpDnaJ具有HPD (HIS-PRO-ASP) motif可與DnaK ATP binding domain 鍵結。將MpgrpE異源表現於grpE缺陷株E. coli DA16中,在高溫逆境固態培養下生長,顯示表現MpgrpE的轉型株生長情形比宿主和表現載體的轉型株佳,顯示MpgrpE可互補EcgrpE的功能。並以北方墨點法分析顯示,grpE-dnaK-dnaJ在熱逆境41 ℃和45 ℃誘導下會分別提高5.9和6.4倍基因轉錄量。由以上結果推論,KJE系統在高溫逆境下會被誘導表現,並具有幫助蛋白修復的能力,所以有利於高鹽甲烷太古生物適應及生存在高溫的環境。
Cellular protein loses their function and structure when encountering the abiotic stress. Heat shock protein DnaK belongs to molecular chaperone and cooperates with its co-chaperone DnaJ and nucleotide exchanger GrpE. DnaK system also co-operate with these two molecular chaperone, chaperonin GroEL/GroES and ClpB belongs to the members of AAA+ protein superfamily. These three systems constitute the ATP-dependent folding function of both nascent polypeptides as well as the salvage of stress-denatured proteins. DnaK system is highly conserved and ubiquitous in eukaryotes and bacteria, but absences in some archaea and little is known about it. A 3.6 kb gene cluster arranged in order grpE-dnaK-dnaJ was cloned from Methanohalophilus portucalensis FDF1T by PCR and Southern blot. RT-PCR and Northern blot results indicated grpE-dnaK-dnaJ was transcribed into the same transcriptional unit. The amino acid sequences analysis of MpDnaK showed the conserved ATP binding domain (EXXKXXXXS) and substrate binding sites (residues L366, V368, T369, L371, A387, L458). MpDnaJ showed the ATPase binding motif HPD (HIS-PRO-ASP) and Zing binding motif (CXXCXGXG) which belongs to type I conserved motif. In vivo analyses indicated MpgrpE could rescued E. coli mutants lacking grpE gene. Northern blot results indicated the relative transcriptional levels were up-regulated by thermal stress. In coclusion, the DnaK system should be beneficial for the adaptative and survival of halophilic methanoarchaea that grown at the salt and temperature harsh solar saltern habitat.
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