Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21695
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dc.contributor.advisor賴美津zh_TW
dc.contributor.advisorMei-Chin Laien_US
dc.contributor.author林珀丞zh_TW
dc.contributor.authorLin, Po-Chenen_US
dc.date2004zh_TW
dc.date.accessioned2014-06-06T07:16:20Z-
dc.date.available2014-06-06T07:16:20Z-
dc.identifier.urihttp://hdl.handle.net/11455/21695-
dc.description.abstract中度嗜鹽甲烷古生菌Methanohalophilus mahii SLP的質體pML,經過定序確認全長為2158 bp,其中最長的ORF (orf1)所轉譯出的胺基酸序列 (328 aa),和高鹽古生菌Halobacterium sp.質體pGRB1的滾環型複製 (RCR)蛋白質 (Rep)有34 %的相似性。ORF1序列上也有Rep蛋白質所共有的三個序列保留區 (motif),根據第三個保留區上的tyrosine數目,pML應歸屬在滾環型複製superfamily I。利用南方墨漬法探測到單股的pML,證實pML是以滾環型的方式進行複製。由M. mahii 的全細胞RNA所做的反轉錄聚合酶連鎖反應 (RT-PCR),亦增幅出和orf1一樣大小的DNA片段 (984 bp),將orf1接到表現載體後送到大腸桿菌中也能轉譯出和預測大小相同的胜肽鏈 (38 kDa)。針對表現蛋白質所做的N端胺基酸定序,確認此表現蛋白質的N端胺基酸序列和預測的ORF1 N端胺基酸序列吻合。藉由anti-His (C-term)-HRP抗體對表現蛋白質做西方墨漬法分析的結果顯示, orf1表現蛋白質的C端具有轉譯後額外添加的組胺酸 (Histidine)可被抗體結合。具有Rep蛋白質辨識切位的pMM3和此大量表現的蛋白質作用後,使pMM3被S1核酸水解酶水解,顯示此蛋白質具有Rep蛋白質能辨識核酸序列並造成切口的活性。將已知的古生菌Rep蛋白質胺基酸序列依其相似程度做系統演化分析,嗜鹽甲烷古生菌質體pML上的Rep蛋白質胺基酸序列和在高鹽環境的極端高鹽古生菌質體pGRB1的相似性遠高於其同源的甲烷菌。zh_TW
dc.description.abstractPlasmid pML (2158 bp) from the moderately halophilic methanogenic archaeon- Methanohalophilus mahii SLP was sequenced. The deduced amino acid sequence of the largest ORF (328 aa) shows 34 % similarity to the putative replication protein (Rep) of pGRB1 from Halobacterium sp. and contains three sequence motifs that conserved in the Rep proteins of rolling circle replication (RCR) mechanism. Based on the numbers of tyrosine residues in motif 3, pML belongs to the superfamily I of RCR. The single-stranded intermediate form of pML was detected by southern blotting, which confirms that pML replicate through RCR mechanism. RT-PCR with total M. mahii RNA and primers specific for orf1 amplified an RNA species of 984 bp. DNA encoding ORF1 was cloned into Escherichia coli and a polypeptide with expected molecular mass of 38 kDa was expressed. Both the N-terminal amino acid sequence and western blotting with anti-His (C-term)-HRP antibody confirm that this overexpressed protein is the translational product of orf1. This protein binds to pMM3 which contains a putative double-stranded replication region and made this fragment sensitive to S1 nuclease indicating the site-specific nuclease activity of this Rep protein. Phylogenetic analysis based on the amino acid sequences of all known archaeal Rep indicated that Rep of pML is clustered to the plasmids of the aerobic extremely halophilic Euryarchaeota, which they inhabited together at hypersaline environment.en_US
dc.description.tableofcontents中文摘要 1 英文摘要 2 表目錄 6 圖目錄 7 壹、前言 8 貳、前人研究 10 一、嗜鹽甲烷古生菌 10 二、古生菌質體 13 三、質體複製機制 14 四、質體的滾環型複製機制與特性 17 五、古生菌質體的複製機制 20 六、嗜鹽甲烷古生菌質體 20 參、 材料與方法 23 一、菌種與質體 23 二、甲烷太古生物培養基組成 23 三、厭氧接菌與菌體生長 25 四、大腸桿菌培養基 25 五、大腸桿菌之培養與保存 25 六、質體的抽取與純化 26 七、核酸膠體電泳分析與紀錄 28 八、DNA片段之回收及純化 29 九、核酸純度之鑑定與定量分析 29 十、DNA黏合反應 30 十一、質體的轉形作用 30 十二、質體複製單股核酸中間物質的偵測 (南方墨漬法) 31 十三、利用RT-PCR方法偵測orf1轉錄產物 35 十四、質體pML之orf1 (rep) 基因之選殖與表現 38 十五、表現質體pET21b-rep 的構築 40 十六、全細胞蛋白質之製備 41 十七、蛋白質電泳 (SDS-PAGE) 41 十八、蛋白質轉印及西方墨漬法 43 十九、Rep蛋白質的大量表現與製備細胞萃取液 45 二十、利用Ni-NTA resin回收純化Rep蛋白質 46 二十一、Rep蛋白質活性測試 48 二十二、改善Rep蛋白質形成包涵體的情形 49 二十三、核酸序列分析 51 肆、結果 53 1. 重新定序後的pML序列以及序列分析 53 2. 偵測pML在複製過程中出現的單股DNA 55 3. 反轉錄聚合酶連鎖反應證實rep基因的轉錄 56 4. 質體pET21b-rep選殖及誘導表現 57 5. Rep蛋白質形成包涵體 (inclusion body) 59 6. 改善Rep蛋白質形成包涵體的情形 60 7. Rep蛋白質純化 61 8. Rep蛋白質活性測試 62 9. Rep蛋白質重新摺疊及活性分析 64 10. Rep演化樹圖 65 伍、討論 67 1. 嗜鹽甲烷古生菌M. mahii之質體pML為滾環型複製質體 67 2. pML之rep基因特色 67 3. pML之Rep蛋白質特性 68 4. Rep蛋白質活性分析 69 5. 溫度及誘導劑量與包涵體形成 70 6. 太古生物的基因於大腸桿菌內的表現 71 陸、結論與展望 73 柒、表與圖 74 捌、參考文獻 96zh_TW
dc.language.isoen_USzh_TW
dc.publisher生命科學系zh_TW
dc.subjectArchaeaen_US
dc.subject太古生物zh_TW
dc.subjectrolling circle replication plasmiden_US
dc.subject滾環型複製質體zh_TW
dc.title滾環型複製的嗜鹽甲烷古生菌質體pML之rep基因及其表現蛋白質的分析zh_TW
dc.titleAnalysis of the rep gene and protein in a RCR plasmid pML from the halophilic methanogenic archaeon-Methanohalophilus mahii SLPen_US
dc.typeThesis and Dissertationzh_TW
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