臺灣博碩士論文加值系統

目錄

中文摘要 i
Abstract ii
目錄 iii
圖目錄 vii
表目錄 viii
第一章 緒論 (INTRODUCTION) 1
1.1 Halobacterium salinarum簡介 (Introduction of Halobacterium salinarum) 1
1.2 H. salinarum蛋白質穩態系統 (Protein homeostasis system in H. salinarum) 3
1.3 分子伴侶蛋白質HSP40/DnaJ簡介 (Introduction of chaperone HSP40/DnaJ) 4
1.4熱休克反應調控 (Regulation of heat shock response) 4
1.4.1 細菌的熱休克反應調控 (Regulation of heat shock response in bacteria) 5
1.4.2 真核生物的熱休克反應調控 (Regulation of heat shock response in eukarya) 6
1.4.3 古菌的熱休克反應調控 (Regulation of heat shock response in archaea) 6
第二章 研究動機 7
第三章 實驗材料 (MATERIALS) 8
3.1 細胞株 (Cell lines and plasmids) 8
3.1.1 Halobacterium salinarum細胞株 (H. salinarum strains) 8
3.2 試劑 (Reagents) 8
3.2.1 細胞培養 (Cell culture) 8
3.2.2 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 11
3.2.3 DNA電泳 (DNA electrophoresis) 11
3.2.4 Total RNAs之萃取 (Isolation of total RNAs) 12
3.2.5 即時聚合酶連鎖反應 (RT-qPCR) 12
3.2.6 分離全裂解蛋白質 (Isolation of whole cell lysate) 12
3.2.7 蛋白質定量 (Protein quantification) 13
3.2.8 十二烷基硫酸鈉聚丙醯胺膠體電泳 (SDS-PAGE) 13
3.2.9 胰蛋白酶消化蛋白質樣品 (Trypsin digestion of protein samples) 13
第四章 實驗方法 (METHODS) 14
4.1 菌株及菌株培養條件 (Cell lines and culture conditions) 14
4.1.1 菌株 (Cell lines) 14
4.1.2 菌株培養條件 (Cells culture conditions) 14
4.2. 以菌落聚合酶連鎖反應確認及篩選基因轉型菌株 (Confirmation and screening of transformants strains through colony PCR) 15
4.3 DNA電泳 (DNA electrophoresis) 15
4.4 即時反轉錄聚合酶連鎖反應 (RT-qPCR) 16
4.4.1 萃取RNA (Total RNA extraction) 16
4.4.2 RNA定量 (Quantification of total RNA) 17
4.4.3 RNA電泳 (RNA electrophoresis) 17
4.4.4 以DNaseI 移除RNA檢體中殘留之genomic DNA (Removal of residual genomic DNA in total RNA samples by DNaseI treatment) 18
4.4.5 RNA樣品進行反轉錄反應製造cDNA (Performing reverse transcription reaction on RNA samples to generate cDNA) 18
4.4.6 即時聚合酶連鎖反應 (Real-time PCR) 19
4.5 以RNA-Seq分析transcriptome (Analysis of transcriptome by RNA-Seq) 19
4.6 H. salinarum細胞株生長曲線測量 (Growth curve of H. salinarum) 19
4.7 蛋白質基本分析之蛋白質樣品製備 (Preparation of protein samples for basic protein analysis) 20
4.7.1 收集全細胞裂解液 (Collection of whole cell lysates) 20
4.7.2 蛋白質樣品定量 (Quantification of protein samples) 21
4.7.3 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS polyacrylamide gel electrophoresis, SDS-PAGE) 21
第五章 實驗結果 (RESULTS) 23
5.1 以colony PCR確認H. salinarum之基因轉型株 23
5.2 H. salinarum Δura3, ΔΔdnaJ及基因互補株之生長曲線分析 23
5.3 以RT-qPCR確認蛋白質體學結果中有顯著變化蛋白質之基因表現變化 25
5.4 在基因互補株中以RT-qPCR確認蛋白質體學結果中有顯著變化蛋白質之基因表現變化 26
5.5 以RNA-Seq分析H. salinarum Δura3和ΔΔdnaJ在37OC及49OC下蛋白質穩態相關基因表現變化 27
5.6 以RNA-Seq分析H. salinarum Δura3和ΔΔdnaJ在37OC及49OC下轉錄因子相關基因表現變化 28
第六章 討論 (Discussions) 29
附錄 51
References 53

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