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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林乃君 | |
| dc.contributor.author | Cheng-Ying Liu | en |
| dc.contributor.author | 劉正寅 | zh_TW |
| dc.date.accessioned | 2021-06-16T16:39:08Z | - |
| dc.date.available | 2017-09-25 | |
| dc.date.copyright | 2012-09-25 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-09-24 | |
| dc.identifier.citation | Alfano, J. R. & A. Collmer (1997) The type III (Hrp) secretion pathway of plant pathogenic bacteria: trafficking harpins, Avr proteins, and death. J. Bacteriol, 179, 5655-5662.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63396 | - |
| dc.description.abstract | 細菌為了爭奪生長空間、營養源或寄主,會運用各種方法互相競爭。最近新 定義出來的第六型分泌系統 (Type VI secretion system,T6SS),被證實是許多動 物病原菌用來進行競爭的工具,例如:Pseudomonas aeruginosa PAO1 及 Vibrio cholerae V52 都可藉由 T6SS 分泌功能蛋白以殺死競爭者或抑制其生長。然而, 植物病原菌是否亦會利用 T6SS 來達到競爭優勢而存活下來,仍待進一步研究探 討。Pseudomonas syringae pv. tomato (Pst) DC3000 為模式植物性病原菌之一,其 基因體中具有兩套可能的 T6SS 基因叢集,分別為 HSI-I 及 HSI-II。目前測試過 的條件下,均僅能觀察到 HSI-II 的表現且分泌關鍵構造 (作用) 蛋白 Hcp2。本 研究證明 PstDC3000 能藉由 HSI-II 與其他植物病原細菌或腸道菌進行生長競爭, 且此競爭作用必須仰賴細菌間的接觸 (contact-dependent);另外我們也證明核心 基因 icmF2、clpV2 以及 vasH2 均會影響 HSI-II 的分泌與競爭作用。此外,在細 菌間的競爭作用中,PstDC3000 是以抑制競爭菌生長而使其菌數下降,與大多數 細菌具殺死目標菌的能力不同。在本研究中亦發現,全域調控因子 GacA 會正向 調控 hcp2 及 HSI-II 的表現,故其突變株會有競爭力下降的情形發生;然而,受 到 GacA 調控的群數感應基因 psyI 對 HSI-II 有影響,但 psyR 則沒有相同的調控 能力。另外我們發現,於 Sucrose minimal medium (SMM) 中生長的 PstDC3000, 會在 pH 值升高時降低 Hcp2 的分泌,同時競爭能力會下降。最後,利用隨機突 變的策略,我們初步發現數個 T6SS 相關基因以外、能夠影響 PstDC3000 生長競 爭的基因,包括 PTO2954、avrE1 及 cfa6。本研究為首次對植物病原菌 PstDC3000 細菌間競爭作用進行較為深入的研究,也對日後相關研究提供了一些基礎。 | zh_TW |
| dc.description.abstract | Bacteria often compete against other microbes for living spaces, resources, or host niches. The newly defined type VI secretion system (T6SS) has been confirmed to provide competition abilities to some animal pathogens such as Pseudomonas aeruginosa PAO1 and Vibrio cholerae V52, which may utilize T6SS to secrete effector proteins to either kill or inhibit their competitors in order to grow better. However, in many plant pathogens bearing T6SS gene clusters, whether they would gain advantages in competition by expressing T6SS further investigation. Pseudomonas syringae pv. tomato (Pst) DC3000 is a plant pathogen, it has two putative T6SS gene clusters, named HSI-I and HSI-II. Under the lab conditions been tested, we can only detect the expression of HSI-II and secretion of the key structural effector protein Hcp2. Here we report that PstDC3000 can grow better in a mixed culture with other phytopathogenic bacteria and enterobacteria by expressing HSI-II in a contact-dependent manner. We also confirmed the core components icmF2, clpV2 and vasH2 could affect HSI-II-dependent secretion and interbacterial competition ability. Furthermore, we found that PstDC3000 uses T6SS to suppress the growth of their competitors instead of killing them, different from the mechanism used by some animal pathogens. In this study, we observed that global regulator GacA could positively controls the expression of hcp2 and HSI-II and also interbacterial competition abilities. Instead of GacA, the downstream regulator PsyI could only affect the expression of HSI-II but not competition, whereas the other regulator, PsyR, didn’t have any effect on both promoters. In addition, we found that when growing in sucrose minimal medium (SMM), Hcp2 secretion in PstDC3000 was lower at higher pH, leading to decreased ability in competition. By means of random mutagenesis, we identified several genes, including PTO2954, avrE1, cfa6, PTO1851/1850 and vasH2, whose mutation would cause decreased ability in interbacterial competition of PstDC3000. This is the first report to investigate the competition of PstDC3000, which could provide fundamental knowledge for the relevant researches afterwards. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T16:39:08Z (GMT). No. of bitstreams: 1 ntu-101-R99623006-1.pdf: 14166950 bytes, checksum: 889fe61c133fb869d7dbaf6c1ea16e13 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 中文摘要.............................................................................................................I Abstract...........................................................................................................II
壹、前人研究......................................................................................................1 一、細菌間的競爭................................................................................................1 1、競爭目的.......................................................................................................1 2、競爭模式.......................................................................................................2 3、競爭機制.......................................................................................................2 二、第六型分泌系統 (Type VI secretion system) ..................................................5 1、第六型分泌系統的組成....................................................................................5 2、第六型分泌系統的作用蛋白..............................................................................8 3、第六型分泌系統的作用機制............................................................................10 4、第六型分泌系統的功能..................................................................................11 (1)、致病能力...................................................................................................11 (2)、細菌間的競爭作用.......................................................................................11 (3)、生物膜的形成.............................................................................................12 (4)、共生現象...................................................................................................12 5、第六型分泌系統的調控..................................................................................13 三、Pseudomonas syringae pv. tomato DC3000 與第六型分泌系統....................15 貳、研究目的....................................................................................................18 參、材料與方法.................................................................................................19 一、菌株及其培養條件.......................................................................................19 二、基本分子生物技術.......................................................................................19 1、大腸桿菌質體 DNA 小量純化.........................................................................19 2、電穿孔用 E. coli 勝任細胞 (competent cell) 之製備.........................................19 3、大腸桿菌轉型作用 (Transformation) .............................................................20 4、接合反應.....................................................................................................20 5、RNA 萃取....................................................................................................21 6、RNA 電泳....................................................................................................21 7 、 反 轉 錄 聚 合 酶 連 鎖 反 應 (Reverse transcription-polymerase chain reaction) .........................................................................................................21 三、質體互補菌株建立.......................................................................................22 四、插入染色體 DNA 之互補菌株建立..................................................................22 五、互補菌株基因表現分析.................................................................................23 1、基因及蛋白質表現分析..................................................................................23 2、菌株外泌蛋白試驗.........................................................................................23 3、RNA polymerase α subunit (RpoA) 抗體製備................................................23 4、西方墨點法 (Western blot) ...........................................................................24 六、競爭試驗 (Competition test) ......................................................................25 1、製備帶有 gfp 基因的 PstDC3000 或 E. coli 轉植株.........................................25 2、PstDC3000 之競爭力測試...........................................................................25 3、競爭菌數計數試驗.......................................................................................25 4、接觸依賴性競爭試驗 (Contact-dependent competition assay) ......................26 5、細菌存活率試驗...........................................................................................26 七、GUS 報導系統活性測試 (GUS activity assay) ...............................................27 八、建立競爭缺陷之隨機突變株 (Random mutagenesis) .....................................28 九、染色體 DNA 抽取純化.................................................................................28 十、突變基因片段分離純化.................................................................................29 十一、突變基因鑒別定序....................................................................................29 肆、結果..........................................................................................................30 一、刪除 PstDC3000 的 HSI-II 會造成 Hcp2 無法表現.........................................30 二、第六型分泌系統的表現有助於 PstDC3000 在與革蘭氏陰性及陽性菌共存 時有較好的活性.............................................................................................................30 三、第六型分泌系統的競爭需仰賴接觸.................................................................33 四、其他核心基因對競爭作用同樣重要.................................................................35 五、PstDC3000 藉由抑制其他細菌生長來達到競爭優勢........................................36 六、感應激酶 (sensor kinase)、全域調控因子 (global regulator) 以及群數感應 (quorum sensing) 對 PstDC3000 第六型分泌系統功能的影響..............................37 七、pH 值會影響 PstDC3000 第六型分泌系統與競爭............................................39 八、影響 PstDC3000 競爭力的其他基因..............................................................40 伍、討論..........................................................................................................43 一、PstDC3000 競爭者篩選...............................................................................43 二、第六型分泌系統參與 PstDC3000 的生長競爭作用..........................................44 三、PstDC3000 利用接觸進行抑制目標的競爭.....................................................46 四、PstDC3000 利用未知蛋白抑制目標菌...........................................................46 五、感應激酶、全域調控因子以及群數感應對 T6SS 的影響....................................47 六、pH 值對第六型分泌系統與競爭力的影響........................................................49 七、PTO2954、avrE1 及 cfa6 與競爭的關聯.......................................................50 陸、參考文獻...................................................................................................53 圖表目錄 Table 1. Bacterial strains and plasmids used in this study...............................62 Table 2. Primers used for semi-quantitative RT-PCR analysis..........................65 Table 3. Primers used for plasmid construction and gene sequencing.............67 Table 4. Genes affect competition ability........................................................69 圖一、 PstDC3000 之 HSI 基因叢集。 ..............................................................70 圖二、 圖二、利用西方墨點法分析 PstDC3000 Hcp2 蛋白於第六型分泌系統突 變株的表現及外泌情形 ..............................................................................................71 圖三、 第六型基因叢集與 PstDC3000 對革蘭氏陽性與陰性菌之生長競爭有關.........72 圖四、 PstDC3000 之第六型基因叢集 HSI-II 對與其他細菌的生長競爭作用 (螢 光訊號) 中是必須的......................................................................................................73 圖五、 PstDC3000 之第六型基因叢集 HSI-II 對與其他細菌的生長競爭作用 (competitive index) 中是必須的.......................................................................74 圖六、 PstDC3000 藉由第六型分泌系統來提高本身的競爭能力必須仰賴與其他 菌的接觸..................................................................................................................75 圖七、 PstDC3000 藉由第六型分泌系統來提高與 Pseudomonas syringae pv. syringae B728a 競爭的生長能力必須仰賴接觸...................................................76 圖八、 PstDC3000 藉由第六型分泌系統來提高與 Erwinia chrysanthemi CAS9 競 爭的生長能力必須仰賴接觸...................................................................................77 圖九、 PstDC3000 藉由第六型分泌系統來提高與 Erwinia chrysanthemi CAS11 競爭的生長能力必須仰賴接觸...................................................................................78 圖十、 PstDC3000 藉由第六型分泌系統來提高與 E. coli BL21(DE3) 競爭的生 長能力必須仰賴接觸...................................................................................................79 圖十一、 預測之第六型分泌系統核心基因 icmF2、vasH2 以及 clpV2 會影響 Hcp2 蛋白之表現或外泌................................................................................................80 圖十二、 icmF2、clpV2 及 HSI-II 突變均會造成 PstDC3000 對 E. coli MG165 的 競爭能力下降.......................................................................................................81 圖十三、 icmF2、clpV2、vasH2 突變後降低之 PstDC3000 和 E. coli MG1655 的 競爭能力可在回復株中恢復....................................................................................82 圖十四、 PstDC3000 的第六型分泌系統 hcp2、icmF2、clpV2 突變株與 E. coli MG1655 共培養後之細菌菌數及存活率................................................................83 圖十五、 E. coli MG1655 與 PstDC3000 的第六型分泌系統 hcp2、icmF2、clpV2 突變株供培養後之細菌菌數以及存活率....................................................................84 圖十六、 PstDC3000 的第六型分泌系統 hcp2、icmF2、clpV2 突變株與 E. coli BL21(DE3) 共培養後之細菌菌數及存活率.............................................................85 圖十七、 E. coli BL21(DE3) 與 PstDC3000 的第六型分泌系統 hcp2、icmF2、clpV2 突變株供培養後之細菌菌數以及存活率.................................................................86 圖十八、 利用半定量 RT-PCR 檢測 PstDC3000 中感應激酶、全域調控因子、群 數感應相關基因對於第六型分泌系統基因表現情形以及與 E. coli MG1655 共培養後之生長情形....................................................................................................................87 圖十九、 全域調控因子 GacA 會對 PstDC3000 第六型分泌系統與競爭能力造成 影響....................................................................................................................88 圖二十、 利用 GUS 報導基因檢測不同生長時期下 PstDC3000 野生株、gacA 突 變株與互補株 gacA:gacA 之 hcp2 表現情形..................................................................89 圖二十一、 利用 GUS 報導基因檢測不同生長時期下 PstDC3000 野生株、gacA 突變株與互補株gacA:gacA 之HSI-II表現情形.................................................................90 圖二十二、 ΔgacA 突變株中 psyI 及 psyR 的表現情形...........................................91 圖二十三、 以西方墨點法分析群數感應調控因子及 sigma-38 因子對 Hcp2 表現 與外泌之影響....................................................................................................92 圖二十四、 利用 GUS 報導基因檢測不同生長時期下 PstDC3000 野生株以及群數 感應調控因子突變株 hcp2 表現情形....................................................................93 圖二十五、 利用 GUS 報導基因檢測不同生長時期下 PstDC3000 野生株以及群數 感應調控因子突變株 HSI-II 表現情形...................................................................94 圖二十六、 利用西方墨點法分析 PstDC3000 於不同 pH 值之 M9 medium、sucrose minimal medium 中 Hcp2 表現與外泌情形.........................................................95 圖二十七、 利用半定量 RT-PCR 分析 PstDC3000 第六型分泌系統核心基因於不 同 pH 值之 Sucrose minimal medium 中之表現情形...........................................96 圖二十八、 培養基 pH 值會影響 E. coli MG1655 與 PstDC3000 共同培養時的生 長能力.............................................................................................................97 圖二十九、 以半定量 RT-PCR 檢測藉由隨機突變所篩選出 PstDC3000 中具影響 競爭能力的基因 avrE1、cfa6 和 PSPTO2954 之表現情形......................................98 圖三十、 以半定量 RT-PCR 檢測 PstDC3000 之 PSPTO2954 突變株與插入染色 體或質體互補之互補菌株的 PSPTO2954 基因表現情形...........................................99 圖三十一、 PSPTO2954 會影響 PstDC3000 的競爭作用.....................................100 圖三十二、 PSPTO2954 不會影響 PstDC3000 中第六型分泌系統核心基因的表現..101 圖三十三、 PSPTO2954 不會影響 PstDC3000 中 Hcp2 的表現及外泌情形...........102 圖三十四、 以半定量 RT-PCR 檢測 avrE1 於 PstDC3000 中的表現情形...............103 圖三十五、 第三型分泌系統對 PstDC3000 的競爭力沒有影響..............................104 附錄 Figure A1、 Schematic representation of a T6SS...........................................105 | |
| dc.language.iso | zh-TW | |
| dc.subject | 第六型分泌系統 | zh_TW |
| dc.subject | 全域調控因子 | zh_TW |
| dc.subject | 細菌間競爭能力 | zh_TW |
| dc.subject | type VI secretion system | en |
| dc.subject | interbacterial competition ability | en |
| dc.subject | global regulator | en |
| dc.title | Pseudomonas syringae pv. tomato DC3000 生長競爭之研究 | zh_TW |
| dc.title | The study of the growth competition of Pseudomonas syringae pv. tomato DC3000 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張晃猷,鄧文玲,吳蕙芬 | |
| dc.subject.keyword | 第六型分泌系統,全域調控因子,細菌間競爭能力, | zh_TW |
| dc.subject.keyword | type VI secretion system,global regulator,interbacterial competition ability, | en |
| dc.relation.page | 105 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-09-24 | |
| 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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