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研究生:林芸
研究生(外文):Yun Lin
論文名稱:高鹽太古生物在溫度、鹽及氧氣逆境下化學伴護因子對分子伴護蛋白基因表現的影響
論文名稱(外文):The relationship of chemical chaperone betaine and molecular chaperone gene expression in halophilic methanogenic Methanohalophilus portucalensis under salt, temperature and oxygen stress
指導教授:賴美津
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:88
中文關鍵詞:化學伴護因子分子伴護蛋白
外文關鍵詞:chemical chaperonemolecular chaperone
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細胞內的環境會受胞外環境的波動變化而在瞬間劇烈的改變,使胞內蛋白因逆境失去正常的功能及結構。分子伴護蛋白系統是生物細胞用來修復失活蛋白的最佳工具,ClpB、DnaK/DnaJ/GrpE(KJE)、GroEL/ES這些分子伴護蛋白系統主要的功能是能將逆境下產生的聚集蛋白進行去凝集化,協助失活蛋白重新折疊後能恢復正常功能。當生物面臨高滲透壓逆境時,會藉由自體生合成或由外界攝取的方式在細胞內累積相容質來對抗逆境,相容質因具有協助蛋白維持正常構形,及協助未折疊多胜肽鏈折疊的功能,被稱為化學伴護因子。高鹽甲烷太古生物Methanohalophilus portucalensis FDF1生長的鹽濃度在1.2到2.9 M間,適應高鹽環境的機制主要為自體生合成β-glutamine、Nε-acetyl-β-lysine與glycine betaine,或自外界攝取glycine betaine及其前驅物,也具有分子伴護蛋白基因clpB及groEL/S,基因表現量受到鹽度及溫度的影響。本研究以北方墨點法分析,clpB基因會在M. portucalensis FDF1生長初期、對數期、及平穩期表現,groELS基因則僅在生長初期與對數期表現,推測GroEL/ES在M. portucalensis FDF1快速生長時協助胞內蛋白結構的穩定,而ClpB在細胞生長時皆會表現。ClpB和GroEL/ES的基因都受高溫熱休克誘導表現,clpB基因表現量在高溫逆境41及45℃時分別增加9.7與8.7倍;groELS基因表現量則分別增加6.7及9.3倍,但clpB與groELS基因表現量皆不受低溫逆境20與29℃的影響。低鹽逆境0.9 M及高鹽逆境3.3 M下,clpB基因表現量分別增加2.04與1.64倍,顯示clpB基因受鹽逆境滲透壓力誘導表現;groELS基因表現量則不受到高鹽逆境3.3、2.7 M及低鹽逆境1.5 M的影響,但在低鹽逆境0.9 M時基因表現量增加1.96倍;推測groELS並不受鹽濃度改變所形成的滲透逆境誘導表現,但胞外鹽濃度太低造成此高鹽生物的新合成蛋白的折疊不正常會誘導groELS基因表現。於鹽及溫度逆境時,在培養液中添加0.5 mM的glycine betaine使3.3 M高鹽逆境時clpB基因表現量下降,高溫逆境45℃時clpB與groELS基因表現量下降,推測M. portucalensis FDF1自培養液攝取glycine betiane作為化學伴護因子,使胞內蛋白結構穩定,便不需大量轉錄、轉譯製造ClpB與GroEL/ES蛋白。絕對厭氧的M. portucalensis FDF1面對氧氣逆境時,clpB基因轉錄量會下降,groELS基因轉錄量則在曝氧20分鐘後開始下降,推測M. portucalensis FDF1因接觸氧氣而逐漸死亡。
Environmental fluctuations usually affect protein structures and functions in cell. By use of molecular chaperone systems, native form proteins are maintained. ClpB, GroEL/ES, and DnaK, DnaJ, GrpE (KJE) chaperone systems are involved in protein disaggregation process. These chaperones liberate the polypeptide from aggregates and facilitate the polypeptide folding correctly. Under high osmotic stress, organism accumulates osmolytes to adapt the stress. Osmolytes are also called chemical chaperones. Not only chemical chaperones help proteins maintain native form, assist refolding of unfolding polypeptide. Methanohalophilus portucalensis FDF1 can grow over a range of external NaCl concentration from 1.2 to 2.9 M. It can de novo synthesize glycine betaine, β-glutamine, and Nε-acetyl-β-lysine as omolytes. It can also transport glycine betaine or precursor of it in cell. M. portucalensis FDF1 has clpB, groEL and groES genes, induced by salt and high temperature stress. At different growth phases, clpB gene transcribed at the lag, exponential and stationary phases and groELS gene only transcribed at the lag and exponential phases. These results suggested that ClpB and GroEL/ES control protein quality during the cell growth. Under hyper salt (3.3 M) stress and hypo salt (0.9 M) stress, clpB transcript levels increased 1.64 and 2.04 fold, respectively. GroELS transcript level increased 1.96 fold under hypo salt (0.9 M) stress, but not in hyper salt (3.3 M) stress. These results suggested that groELS gene expression was not induced by salt and osmotic stress. However, the low salt concentrations for high salt lover might lead to the increasing proteins folding and aggregation which induced the groELS gene expression. Under heat stress, clpB transcript levels increased 9.7 or 8.7 fold under 41 or 45℃ and groELS transcript levels increased 6.7 or 9.3 fold, and both genes have no effect on cold stress. With the addition of glycine betaine, clpB transcript levels decreased both at hyper salt and heat stress and groELS transcript levels decreased under heat stress. These effects indicated glycine betaine may function as chemical chaperone to protect the protein.
中文摘要...................................................Ⅰ
英文摘要...................................................Ⅱ
圖目錄.....................................................Ⅴ
壹、前言....................................................1
貳、前人研究................................................3
ㄧ、逆境對生物體內蛋白質及酵素的影響......................3
(一)酸鹼值(pH).......................................3
(二)溫度(Temperature)...............................4
(三)高鹽(High salt)..................................4
(四)氧化反應(Oxidation).............................4
二、分子伴護蛋白(molecular chaperone)...................5
(一)ClpB/Hsp100.......................................5
(二)DnaK、DnaJ、GrpE/Hsp70 chaperone machine..........6
(三)Chaperonin........................................7
三、化學伴護因子..........................................8
四、化學伴護因子與分子伴護蛋白的關係......................9
五、甲烷太古生物Methanohalophilus portucalensis滲透壓的適應與調節.....................................................13
參、材料與方法.............................................13
ㄧ、菌種.................................................13
二、 配置含12%NaCl的無氧 H-P培養液.......................13
三、配置還原劑與碳源.....................................14
四、厭氧接菌.............................................14
五、生長曲線測定.........................................15
六、RNA萃取..............................................15
七、RNA定量..............................................15
八、質體抽取與純化.......................................16
九、聚合酶連鎖反應(Polymerase Chain Reaction)..........17
十、PCR產物回收與純化....................................17
十ㄧ、北方墨點法.........................................17
(1)探針製備..........................................18
(2)探針標定效率測試..................................18
(3)配置0.1% diethylpyrocarbonate溶液(DEPC-water)...19
(4)配置10X MOPS buffer...............................19
(5)RNA實驗中的器具處理...............................19
(6)RNA電泳、轉漬與固定...............................19
(7)雜合反應(hybridiazition)........................20
(8)轉漬膜洗滌(Stringency wash).....................20
(9)免疫偵測(Immunological detection)...............20
十二、計算基因相對轉錄量(relative transcription level).21
十三、clpB、groELS、mcr於不同生長時期基因表現量測定實驗..21
十四、鹽(NaCl)逆境(salt stress)......................21
十五、溫度逆境(temperature stress).....................22
十六、氧氣逆境(oxygen stress)..........................22
十七、mcrA(methyl-coenzyme M reductase)部分基因選殖....22
(1)引子設計..........................................22
(2)聚合酶連鎖反應增幅mcrA部分基因....................22
(3)DNA黏合反應.......................................23
(4)勝任細胞製備......................................23
(5)質體轉型作用(transformation)....................23
(6)核酸定序及序列分析................................23
肆、結果...................................................25
ㄧ、不同生長時期clpB、groELS基因表現量測定實驗...........25
二、鹽逆境與化學伴護因子甜菜鹼對clpB、groELS基因表現量的影響.........................................................25
三、溫度逆境與化學伴護因子甜菜鹼對clpB、groELS基因表現量的影響.......................................................25
四、氧氣逆境對clpB、groELS基因表現量的影響...............26
五、以聚合酶連鎖反應增幅Methanohalophilus portucalensis FDF1的mcrA基因片段.........................................26
六、不同生長時期mcr基因表現量測定實驗....................27
七、鹽逆境與化學伴護因子甜菜鹼對mcr基因表現量的影響......27
八、溫度逆境與化學伴護因子甜菜鹼對mcr基因表現量的影響....27
九、氧氣逆境對mcr基因表現量的影響........................28
伍、討論...................................................28
ㄧ、不同生長時期clpB、groELS基因表現量...................29
二、鹽逆境與化學伴護因子甜菜鹼對clpB、groELS基因表現量的影響.........................................................29
三、溫度逆境與化學伴護因子甜菜鹼對clpB、groELS基因表現量的影響.......................................................31
五、氧氣逆境...............................................32
六、不同逆境下mcr基因表現量之探討..........................33
陸、結論與未來展望.........................................34
柒、圖.....................................................36
捌、參考文獻...............................................51
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