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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳蕙芬 | |
| dc.contributor.author | Yu-Feng Chan | en |
| dc.contributor.author | 湛玉鳳 | zh_TW |
| dc.date.accessioned | 2021-06-15T05:51:05Z | - |
| dc.date.available | 2015-08-20 | |
| dc.date.copyright | 2010-08-20 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-18 | |
| dc.identifier.citation | Berlett, B. S. & Stadtman, E. R. (1997). Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272: 20313-20316.
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Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413: 852-856. Miller, S. T., Xavier, K. B., Campagna, S. R., Taga, M. E., Semmelhack, M. F., Bassler, B. L. & Hughson, F. M. (2004). Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2. Mol Cell 15: 677-687. Nahvi, A., Barrick, J. E. & Breaker, R. R. (2004). Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes. Nucleic Acids Res 32: 143-150. Nahvi, A., Sudarsan, N., Ebert, M. S., Zou, X., Brown, K. L. & Breaker, R. R. (2002). Genetic control by a metabolite binding mRNA. Chem Biol 9: 1043. Roy, M. F. & Malo, D. (2002). Genetic regulation of host responses to Salmonella infection in mice. Genes Immun 3: 381-393. Schulte, L. L., Stauffer, L. T. & Stauffer, G. V. (1984). Cloning and characterization of the Salmonella typhimurium metE gene. J Bacteriol 158: 928-933. Simons, R. W., Houman, F. & Kleckner, N. (1987). Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53: 85-96. Stadtman, E. R. & Berlett, B. S. (1998). Reactive oxygen-mediated protein oxidation in aging and disease. Drug Metab Rev 30: 225-243. Sudarsan, N., Hammond, M. C., Block, K. F., Welz, R., Barrick, J. E., Roth, A. & Breaker, R. R. (2006). Tandem riboswitch architectures exhibit complex gene control functions. Science 314: 300-304. Urbanowski, M. L. & Stauffer, G. V. (1989). Role of homocysteine in metR-mediated activation of the metE and metH genes in Salmonella typhimurium and Escherichia coli. J Bacteriol 171: 3277-3281. Valentin-Hansen, P., Eriksen, M. & Udesen, C. (2004). The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Microbiol 51: 1525-1533. Wang, J. X., Lee, E. R., Morales, D. R., Lim, J. & Breaker, R. R. (2008). Riboswitches that sense S-adenosylhomocysteine and activate genes involved in coenzyme recycling. Mol Cell 29: 691-702. Waters, L. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47214 | - |
| dc.description.abstract | 腸道沙門氏菌為世界性之食物媒介疾病，其中Salmonella Typhimurium LT2 菌株之基因組更已確定，與模式細菌大腸桿菌之基因組具高相似性，且對人類之毒性較模式動物小鼠相對為低，可作為研究流行病學病原菌之相對應的模式微生物。也可藉由生理調控機制之研究，以對其更加了解。其中，甲硫胺酸之合成為細菌生存所必需，於合成路徑最後一步之作用酵素MetE 更是合成過程中所必須的重要酵素之一。
因此，於本研究將建構不同之缺失株及相關的系列選殖質體，利用西方墨點法及β-galactosidase 活性分析等不同的基因表現方法，探討革蘭氏陰性菌之沙門氏菌的metE 基因調控情形，並確定其調控是否與核糖開關或小分子RNA FnrS 相關。 結果發現metE mRNA 5’端未轉譯區域的+30~+68 片段缺失會造成metE 的表現量明顯下降，可知metE 5’端未轉譯區域對調控的必要性。並且，metE 之表現量下降差異非受抑制因子MetJ 之影響。而革蘭氏陰性菌腸道沙門氏菌之metE 基因調控機制與革蘭氏陽性菌克勞氏芽孢桿菌的核糖開關之調控機制不同。另外，也發現Hfq 及FnrS 與metE 之表現具有相關性，且小分子RNA FnrS 的大量表現可抑制metE 之表現，與metE mRNA 5’端未轉譯區域可為互補與接合。而兩者任一之結構的缺失皆會降低其對metE 基因表現之影響。 希望透過對此腸道沙門氏菌metE 調控機制之了解，除可更加認識腸道沙門氏菌metE 基因之調控機制外，也期將具可利用性之調控機制應用於其他相類似基因之研究上。 | zh_TW |
| dc.description.abstract | Salmonella enterica is one of the pathogenic bacteria that cause the worldwide food-borne desease. One strain, Salmonella Typhimurium LT2, its genome has been sequenced with a similarity to Escherichia coli. Salmonella enterica is more toxicity in mice than in human, and it can be used for an epidemiological study of human pathogens. However, Salmonella Typhimurium LT2, itself, can be used as a model microorganism for its physiological study. Methionine is an essential amino acid for the bacteria, and in the methionine biosynthesis, the last step was modulated by MetE, which is a key enzyme in this biosynthetic pathway.
Here, a set of deletion mutations in metE leader sequences were made; using Western blotting and the assays of β-galactosidase acitivity to analyze metE gene expression. In addition, we also tested whether it is related to the regulation of riboswitch or is controlled by small RNA FnrS. Our results showed that a metE 5’ untranslated region with bp +30 to +68 deletion causes the metE gene expression decrease. Therefore, metE 5’ untranslated region is necessary for its gene expsresion, and this decrease is not affected by repressor MetJ. Howerer, the mechanism was different from the riboswitch of Bacillus clause. In addition, we also found that Hfq and FnrS are related to metE expression, and an v overexpression of FnrS inhibits the metE expression. The distortion of any secondary structure of fnrS or metE gene, has an effect on the expression of metE. Through our study, the regulation of Salmonella enterica metE gene were extensively studied and it would be very helpful for understanding of the regulation of other gene expression, which have a similar regulatory mechanism. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T05:51:05Z (GMT). No. of bitstreams: 1 ntu-99-R97623025-1.pdf: 5387556 bytes, checksum: ee6b86772a4867f9f48defda29710d32 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 目錄
口試委員會審定書………………………………………………………………………i 誌謝……………………………………………………………………………………...ii 摘要……………………………………………………………………………………..iii Abstract………………………………………………………………………………….iv 目錄……………………………………………………………………………………..vi 表目錄…………………………………………………………………………………viii 圖目錄…………………………………………………………………………..ix 附圖附錄………………………………………………………………………………x 一、前言…………………………………………………………………….1 1.1 腸道沙門氏菌之簡介…………………………………………………………..1 1.2 甲硫胺酸之簡介………………………………………………………………2 1.3 MetE之簡介…………………………………………………………………...5 1.4 基因表現之調控因子…………………………………………………………6 1.5 FnrS之簡介……………………………………………………………………9 1.6 研究動機與目的………………………………………………………………10 二、材料與方法…………………………………………………………..11 2.1 實驗材料………………………………………………………………………11 2.2 一般性實驗方法………………………………………………………………15 2.3 選殖基因表現系統之建立……………………………………………………19 2.4 噬菌體一般實驗方法…………………………………………………………22 2.5 基因剔除 (knockout) 之突變株建構………………………………………23 2.6 PmetE::lacZ fusion之菌體建構………………………………………………25 2.7 蛋白質誘導表現分析…………………………………………………………32 三、結果…………………………………………………………………..38 3.1 選殖質體及突變菌株之建構…………………………………………………38 3.2 metE選殖質體對metE突變菌株的生長影響……………………………39 3.3 核糖開關相關之代謝分子對metE表現的影響……………………………40 3.4 不同長度之5’端未轉譯區域對metE表現的影響…………………………41 3.5 5’端未轉譯區域之不同部位對metE::lacZ表現的影響……………………42 3.6 抑制因子MetJ對metE::lacZ表現的影響……………………………………43 3.7 FnrS及Hfq對metE表現的影響……………………………………………43 3.8 FnrS之不同區域對metE表現的影響………………………………………44 四、討論…………………………………………………………………..46 4.1 metE 5’端未轉譯區域與核糖開關之相關性…………………………………46 4.2 metE 5’端未轉譯區域與metE之調控的相關性……………………………..47 4.3 metE::lacZ於轉錄及轉譯時表現差異的意義………………………………47 4.4 抑制因子MetJ與metE 5’端未轉譯區域之相關性…………………………..48 4.5 FnrS及Hfq與metE表現調控之相關性……………………………………48 4.6 FnrS與metE 5’端未轉譯區域之調控的相關性……………………………...49 五、結論……………………………………………………………..50 六、參考文獻……………………………………………………………..51 表目錄 表1、本論文所使用之菌株……………………………………………………………56 表2、本論文所使用之質體及噬菌體…………………………………………………58 表3、本論文所使用之引子對…………………………………………………………59 圖目錄 圖1、甲硫胺酸之合成路徑及S-腺苷甲硫胺酸之代謝循環路徑…………………...61 圖2、不同長度的5’端未轉譯區之metE基因建構…………………………………62 圖3、metE選殖質體之生長測試…………………………………………………….63 圖4、代謝分子對metE表現的影響…………………………………………………64 圖5、MetE之質譜比對結果………………………………………………………….65 圖6、不同長度之5’端未轉譯區域對MetE表現的影響……………………………66 圖7、不同長度之5’端未轉譯區域及HA標記對MetE表現的影響………………67 圖8、metE-lacZ融合基因之建構…………….............................................................68 圖9、不同長度的轉錄及轉譯融合基因metE::lacZ之活性………………………..69 圖10、抑制因子MetJ之缺失對metE 5’端未轉譯區域調控表現之影響…………70 圖11、FnrS及Hfq缺失對metE表現量之影響……………………………………71 圖12、FnrS對metE表現之影響……………………………………………………..72 圖13、不同內部缺失之metE 5’端未轉譯區域RNA二級結構的預測…………….73 附圖目錄 附圖1、FnrS結構圖…………………………………………………………………74 附圖2、大腸桿菌metE mRNA與FnrS之序列比對及FnrS結構預測…………75 | |
| dc.language.iso | zh-TW | |
| dc.title | 沙門氏菌metE基因之表現調控 | zh_TW |
| dc.title | Regulation of Salmonella Typhimurium metE Gene Expression | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 邱志郁,謝文陽,陳建德,劉俊民 | |
| dc.subject.keyword | 腸道沙門氏菌,甲硫胺酸,維生素B12非依賴型甲硫胺酸合成酶,MetE,5’端未轉譯區域,小分子RNA, | zh_TW |
| dc.subject.keyword | Salmonella enterica,methionine,MetE,5’ untranslated region,small RNA, | en |
| dc.relation.page | 75 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-18 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| 顯示於系所單位： | 農業化學系 | |
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