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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林乃君 | |
dc.contributor.author | Nai-Tan Hsu | en |
dc.contributor.author | 許乃丹 | zh_TW |
dc.date.accessioned | 2021-06-15T02:59:11Z | - |
dc.date.available | 2021-08-16 | |
dc.date.copyright | 2011-08-23 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-16 | |
dc.identifier.citation | Akeda, Y., and Gala´n, J.E. 2005. Chaperone release and unfolding of substrates in type III secretion. Nature 437:911-915.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44464 | - |
dc.description.abstract | 革蘭氏陰性菌具雙層膜,因而發展出多種分泌系統,其中第一型到第五型已被廣泛研究,第六型分泌系統 (Type VI secretion system, T6SS) 則是近年來才被確認之新型分泌系統,已被證實和病原菌的致病性有關。Pseudomonas syringae pv. tomato DC3000 (PstDC3000) 是一種番茄病原菌,具有兩套T6SS基因叢集 (gene clusters),其內除了有分泌系統組裝蛋白的基因之外,還包含有具調控T6SS分泌蛋白Hcp功能的sigma 54轉錄活化因子的基因,在其基因體中的編碼分別為PSPTO_2549及PSPTO_5424。本研究透過該二基因突變株,利用GUS報導基因系統來探討其對第六型分泌系統相關基因表現之影響,結果發現PSPTO_5424對於hcp-2的調控扮演極為重要的角色,而PSPTO_2549可能會幫助PSPTO_5424調控hcp-2。此外,利用GUS報導基因系統也找到了在hcp-2上游240bp到360bp之間,具有調控hcp-2表現的重要序列。與第三型分泌系統不同的是,第六型分泌系統在 King’s B 培養基中,只要菌數夠多即可大量表現,不必完全以基本培養基配合 fructose或其他醣類才能誘導其表現。這或許可以解釋為何之前的結果顯示第六型分泌系統不若第三型分泌系統,在 PstDC3000於植物的病原性上具有極重要的功能。 | zh_TW |
dc.description.abstract | Due to the complexity of the double membrane structure, Gram-negative bacteria develop a variety of secretion systems, including Type I to Type V secretion system (T1SS to T5SS ) to secret proteins into the surrounding environments. In 2006, a novel secretion system, termed Type VI secretion system (T6SS), was recognized as another important virulence factor of the bacterial pathogen. In tomato pathogen, Pseudomonas syringae pv. tomato DC3000 (PstDC3000), two T6SS gene clusters in addition to its secretion components genes, hcp1 and hcp2, was identified using in silico analysis. Each cluster contains one copy of vasH homolog (PSPTO_2549 and PSPTO_5424), the gene coding for the sigma 54-dependent transcriptional activator, which controls the expression of T6SS-secreted protein, Hcp. By means of PSPTO_2549 and PSPTO_5424 mutants in accompany with a GUS reporter system, we showed that PSPTO_5424 plays a very important role in regulation of hcp-2 expression and PSPTO_2549 may cooperates with PSPTO_5424 to control the hcp-2 expression. Moreover, we also found that 240bp to 360bp upstream of the hcp-2 ORF is required for hcp-2 expression. Unlike type III secretion systems, genes in the second T6SS gene cluster or hcp-2 were expressed when PstDC3000 were grown in King’s B medium in a cell density-dependent manner, and expression of these genes is not completely induced by sugars as that of type III effector genes. The different expression patterns between T3SS and T6SS may explain why T6SS is not as important as T3SS in the virulence activity of PstDC3000 in planta. | en |
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dc.description.tableofcontents | 目錄
誌謝…………………………………………………………………I 中文摘要……………………………………………………………III Abstract………………………………………………………………IV 壹、前人研究………………………………………………………1 一、革蘭氏陰性菌的分泌系統………………………………1 二、 第六型分泌系統………………………………………3 1. 第六型分泌系統的組成蛋白……………………3 2. 第六型分泌系統之作用蛋白…………………5 3. 第六型分泌系統的組裝和分泌蛋白機制………8 三、 第六型分泌系統的功能………………………………9 1. 致病力 (virulence)……………………………9 2. 對抗捕食 (resisting predation)……………9 3. 共生現象 (symbiosis)………………………9 4. 細菌間的競爭性 (interbaterial competition)…10 5. 生物膜 (biofilm) 之合成…………………………10 6. 感應環境壓力 (stress response)………………11 四、 第六型分泌系統之調控機制……………………………11 五、 Pseudomonas syringae pv. tomato DC3000 (PstDC3000)........13 貳、研究目標………………………………………………………15 參、材料與方法…………………………………………………16 一、 菌株與培養條件……………………………………16 二、 sigma 54 轉錄活化因子的親緣關係分析…………16 三、 基本分子選殖方法……………………………………17 1. 選殖基因片段的製備與純化………………………17 2. 大腸桿菌質體DNA的小量純化……………………17 3. 接合反應 (Ligation)……………………………18 4. 大腸桿菌勝任細胞 (Eletrocompetent cells) 的製備.......18 5. 大腸桿菌的轉型 (Transformation)…....18 四、 PstDC3000突變株之之製備...…………………18 1. 自殺載體的製備………………………………18 2. 接合反應 (Conjugation)……………………19 3. 突變株之篩選…………………………………19 五、 南方墨漬法.................................20 1. 抽取細菌之染色體DNA....................20 2. DNA轉印................................20 3. 探針 (probe) 製備......................21 4. 雜合反應 (Hybridization)...............21 六、 GUS報導基因系統...........................21 1. 建構Pst DC3000 hcp2 啟動子之GUS報導基因系統.21 2. β-葡萄糖苷酸酶 (β-glucuronidase, GUS) 活性之 測量........22 3. 以含X-Gluc的培養基檢測β-葡萄糖苷酸酶 (β-glucuronidase, GUS) 活性...........22 七、 半定量反轉錄聚合酶連鎖反應 (reverse transcription-polymerase chain reaction, RT-PCR)...................23 1. RNA萃取................................23 2. RNA電泳................................24 3. 利用DNase去除RNA樣品中殘留的DNA........24 4. 合成cDNA...............................24 5. 聚合酶連鎖反應.........................25 八、 Hcp2蛋白表現分析..........................25 肆、結果............................................27 一、 PSPTO_2549和PSPTO_5424的特性分析..........27 1. PSPTO_2549和PSPTO_5424的親源演化分析...27 2. PSPTO_2549和PSPTO_5424的胺基酸序列比較.27 二、 突變PSPTO_2549和PSPTO_5424對PstDC3000第六型分泌系統相關基因的影響....................................28 1. PSPTO_2549和PSPTO_5424突變株之確認.....28 2. 突變PSPTO_2549和PSPTO_5424不會影響PstDC3000中t6ss1及 t6ss2基因叢集的表現.........................28 3. 突變PSPTO_2549和PSPTO_5424不會影響hcp-1的表現..................................................29 4. PSPTO_5424缺失會使hcp-2不表現..........30 5. PSPTO_2549和PSPTO_5424突變株之第六型分泌系統相關基因的表現........................................30 三、 hcp-2上游360bp到240bp之間具有調控其表現之重要序列..................................................31 四、 培養基中的醣類對PstDC3000第六型分泌系統相關基因的影響................................................31 1. 於固態培養基上測試,不同的醣類會影響T6SS相關基因的表現............................................31 2. 液態培養基中galactose和mannose對於t6ss1和t6ss2基因叢集的表現有最佳的效果..........................32 3. 液態培養基中galactose對於hcp-1和hcp-2基因的表現有最佳的效果........................................32 五、 不同醣類對Hcp-2的蛋白質表現情形...........33 六、 第六型分泌系統相關基因表現情形............33 伍、討論............................................34 一、 PSPTO_2549和PSPTO_5424對於T6SS相關基因的影響..................................................34 二、 不同醣類對PstDC3000第六型分泌系統相關基因會產生 不同的影響................................36 陸、參考文獻..................................................40 圖表目錄 表一、 本研究中所使用的菌株與質體...................46 表二、 本研究中所使用之引子.........................47 圖一、 不同Pseudomonas spp.中sigma 54 轉錄活化因子之親緣性分析..................................................49 圖二、 PSPTO_2549和PSPTO_5424胺基酸序列比對.........50 圖三、 PstDC3000 PSPTO_2549突變株之構築與確認.......51 圖四、 PstDC3000 PSPTO_5424突變株之構築與確認.......52 圖五、 PSPTO_2549和PSPTO_5424對t6ss1基因叢集表現量之影響..................................................53 圖六、 PSPTO_2549和PSPTO_5424對t6ss2基因叢集表現量之影響..................................................54 圖七、 PSPTO_2549和PSPTO_5424對hcp-1基因叢集表現量之影響..................................................55 圖八、 PSPTO_2549和PSPTO_5424對hcp-2基因叢集表現量之影響..................................................56 圖九、 利用半定量反轉錄聚合酶連鎖反應分析Pst DC3000野生型、Δ2549、Δ5424和Δ2549Δ5424中hcp-2、clpV、和icmF的轉錄情形..................................................57 圖十、 hcp-2上游不同長度片段對GUS活性表現之影響.....58 圖十一、 hcp-1和hcp-2上游360-240bp序列比對..........59 圖十二、 醣類對t6ss1和t6ss2基因叢集及hcp-1和hcp-2表現的影響..................................................60 圖十三、 醣類對t6ss1和t6ss2基因叢集表現影響之定量分析..................................................62 圖十四、醣類對hcp-1和hcp-2基因叢集表現影響之定量分析..................................................63 圖十五、 以含有galactose的minimal medium培養PstDC3000時,細胞中Hcp-2 蛋白質的累積量............................64 圖十六、 以含有glucose的minimal medium培養PstDC3000時,細胞中Hcp-2蛋白質的累積量...............................65 圖十七、 以含有fructose的minimal medium培養PstDC3000時,細胞中Hcp-2蛋白質的累積量...............................66 圖十八、 以含有mannitol的minimal medium培養PstDC3000時,細胞中Hcp-2蛋白質的累積量................................67 圖十九、 以含有sucrose的minimal medium培養PstDC3000時,細胞中Hcp-2蛋白質的累積量................................68 圖二十、 利用半定量反轉錄聚合酶連鎖反應分析Pst DC3000野生株中hcp-2、clpV、 icmF、PSPTO_2549和PSPTO_5424的轉錄情形..69 附錄 附錄一、 利用半定量反轉錄聚合酶連鎖反應分析Pst DC3000野生株中hcp-2 的轉錄情形......................................70 附錄二、 利用半定量反轉錄聚合酶連鎖反應分析Pst DC3000野生株中clpV-1的轉錄情形......................................................71 附錄三、 利用半定量反轉錄聚合酶連鎖反應分析Pst DC3000野生株中clpV-2的轉錄情形......................................................72 附錄四、 利用半定量反轉錄聚合酶連鎖反應分析Pst DC3000野生株中icmF-1的轉錄情形......................................73 附錄五、 利用半定量反轉錄聚合酶連鎖反應分析Pst DC3000野生株中icmF-2的轉錄情形......................................74 附錄六、 利用半定量反轉錄聚合酶連鎖反應分析Pst DC3000野生株中PSPTO_2549的轉錄情形..................................75 附錄七、 利用半定量反轉錄聚合酶連鎖反應分析Pst DC3000野生株中PSPTO_5424的轉錄情形..................................76 | |
dc.language.iso | zh-TW | |
dc.title | Pseudomonas syringae pv. tomato DC3000
第六型分泌系統基因調控之初步探討 | zh_TW |
dc.title | Studies on the regulation of type VI secretion system-related genes in Pseudomonas syringae pv. tomato DC3000 | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧文玲,徐媛曼,鍾嘉綾 | |
dc.subject.keyword | 第六型分泌系統,sigma 54轉錄活化因子,sigma 54因子,Hcp,Pseudomonas syringae pv. tomato DC3000, | zh_TW |
dc.subject.keyword | Type VI secretion system (T6SS),sigma 54-dependent transcriptional activator,sigma 54 factor,Hcp,Pseudomonas syringae pv. tomato DC3000, | en |
dc.relation.page | 76 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-08-16 | |
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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