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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 | Yun-Chen Lee | en |
dc.contributor.author | 李昀蓁 | zh_TW |
dc.date.accessioned | 2021-06-15T00:14:34Z | - |
dc.date.available | 2021-08-15 | |
dc.date.copyright | 2011-08-23 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-15 | |
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Phosphorylation of the Pseudomonas syringae effector AvrPto is required for FLS2/BAK1-independent virulence activity and recognition by tobacco. Plant J 61:16-24. Zhang, J.K., White, A.K., Kuettner, H.C., Boccazzi, P., and Metcalf, W.W. 2002. Directed mutagenesis and plasmid-based complementation in the methanogenic archaeon Methanosarcina acetivorans C2A demonstrated by genetic analysis of proline biosynthesis. J Bacteriol 184:1449-1454. Zhao, Y.F., Thilmony, R., Bender, C.L., Schaller, A., He, S.Y., and Howe, G.A. 2003. Virulence systems of Pseudomonas syringae pv. tomato promote bacterial speck disease in tomato by targeting the jasmonate signaling pathway. Plant Journal 36:485-499. Zheng, J., and Leung, K.Y. 2007. Dissection of a type VI secretion system in Edwardsiella tarda. Mol Microbiol 66:1192-1206. Zusman, T., Feldman, M., Halperin, E., and Segal, G. 2004. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41232 | - |
dc.description.abstract | Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) 為造成番茄細菌性斑點病的植物病原菌,由於其相關的研究頗多,加上其基因體序列已於2003年解序完成,故成為研究植物與病原菌交互作用的模式系統生物。一般來說,為了探討特定基因的功能性,常以刪除 (knock-out) 基因方式來觀察表型 (phenotype) 的變化,接者再以互補方式 (complementation) 確認所觀察到的表型變化確實為該基因所致。在細菌的研究上,常以質體攜帶被刪除基因的方式來建構互補菌株,但已有研究顯示,利用質體來表現互補基因時,其表現量有時並未與野生株相似,或是會有質體穩定性上的問題。為解決此問題,本篇研究以發展能將欲表現基因插入染色體定點位置的新方法為目的,來建構 Pst DC3000 的互補菌株。首先利用生物資訊學分析找出在 Pst DC3000 染色體上可能具有潛力之區域,將該區域以PCR增殖後,選殖入自殺載體中,建構出 pK18msLP,以利用 sacB 基因特性為原理的策略將基因插入預定位置。接著以在 Pst DC3000 上研究頗多的第三型及第六型分泌系統為對象,測試該策略之可行性及互補效果。結果顯示此法可成功建構互補菌株,且在生長競爭試驗中其互補效果較以質體互補佳。另一方面,前人研究提到在植物分析中質體可能有喪失的情形,在本研究中確實也觀察到此現象,攜帶 avrPtoB 基因的質體互補菌株在感染感病品種番茄及菸草後第五天該質體保留率皆下降 12~20% 左右。以目前的結果看來,利用 pK18msLP 確實能將基因成功插入 Pst DC3000 定點的位置上,較利用質體建構的互補菌株更能穩定地存在於細胞中,且互補基因的表現量與功能也較趨近於野生株。 | zh_TW |
dc.description.abstract | Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), the causing agent of tomato speck disease, is a model pathogen for the studies of the molecular mechanisms underlying gene-for-gene resistance and disease susceptibility. Its genome sequence has been completed in 2003; thus, it is important to know the function of each gene in the post genomic era. In general, to explore the function of genes, we often use loss- or gain-of-function strategies. For studying bacteria, complementation of a mutant strain was usually performed using a plasmid-based method. However, some properties of the plasmid, such as multicopy and instability, may hamper the utilization of plasmid to carry the gene for complementation. In order to solve this problem, we developed a new site-specific chromosomal integration strategy for genetic expression in Pst DC3000. First, potential regions suitable for integration of genes in the genome of Pst DC3000 was identified using bioinformatic analysis. Then a sacB-based strategy was applied using a suicide vector pK18msLP to integrate gene-of-interest into the designed region. The efficacy of this method was assessed by using mutants deficient in certain T3SS and T6SS effectors. Our data revealed that the chromosome-integrated method was better than the plasmid-borne method for complementation of hcp1 and hcp2, two T6SS effectors in a growth competition assay. In addition, a plasmid carrying avrPtoB was not stable in Pst DC3000 as we observed 12-20% decrease in the plasmid retention ratio when it was inoculated into the susceptible tomato lines and N. benthamiana. In conclusion, we have successfully established a stable chromosomal integration strategy for gene expression with similar or even better effects than the plasmid-encoded strategy. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T00:14:34Z (GMT). No. of bitstreams: 1 ntu-100-R98623023-1.pdf: 2946600 bytes, checksum: 70f4050e7804be82b7bda34595a34c31 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 誌謝………………………………………………………………………I
中文摘要…………………………………………………………………II Abstract………………………………………………………………III 壹、前人研究……………………………………………………………1 一、Pseudomonas syringae pv. tomato DC3000 (Pst DC3000)……1 二、第三型分泌系統 (Type III secretion system,T3SS)………2 三、第六型分泌系統 (Type VI secretion system,T6SS)…………4 1.第六型分泌系統的組成……………………………………………5 2.Hcp蛋白質………………………………………………………………7 3.第六型分泌系統參與細菌的交互作用………………………………7 四、互補菌株相關研究…………………………………………………8 1.遺傳學研究……………………………………………………………8 2.以質體互補的方式……………………………………………………10 (1)pRK2-based質體……………………………………………………10 (2)pBR-based質體………………………………………………………11 (3)pUC-based質體………………………………………………………11 (4)pCPP45質體…………………………………………………………11 (5)基因為多套組 (multicopy) 形式…………………………………12 (6)質體的不穩定性…………………………………………13 3.將基因插入染色體之互補菌株……………………………………14 (1)跳躍子媒介插入基因之方式………………………………………15 (2)染色體定點插入基因之方式………………………………………16 貳、研究目標……………………………………………………………18 參、材料與方法…………………………………………………………19 一、菌株及其培養條件…………………………………………………19 二、植物及其生長條件…………………………………………………19 三、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………20 3.大腸桿菌轉型作用 (transformation)……………………………20 4.接合反應 (conjugation)…………………………………………20 5.RNA 萃取…………………………………………………………21 6.RNA 電泳…………………………………………………………21 7.反轉錄聚合酶連鎖反應 (reverse transcriptase-polymerase chain reaction, RT-PCR)……………………………………………21 四、質體互補菌株之建立………………………………………………22 五、建立插入染色體之互補菌株………………………………………22 1.找尋染色體中合適的插入基因位置………………………………22 2.建構 pK18msLP……………………………………………………23 3.建立插入染色體互補菌株…………………………………………23 4下游基因 PSPTO_0286 及 PSPTO_0287 的表現分析…………24 六、互補菌株基因表現分析……………………………………………24 1.基因及蛋白質表現分析試驗………………………………………24 2.互補菌株蛋白質外泌試驗…………………………………………24 3.西方墨點法 (Western blotting)…………………………………25 七、競爭試驗 (competitive test)…………………………………25 1.製備帶有 gfp 基因的 E. coli 轉殖株……………………………25 2.Pst DC3000 及 E. coli 競爭力測試………………………………26 3.競爭菌數計數試驗…………………………………………………26 八、質體保留率試驗 (plasmid retention test)…………………26 1.KB 液態培養基中進行質體保留率試驗…………………………27 2.HrpMM 中進行質體保留率試驗…………………………………27 3.植物體中質體保留率試驗…………………………………………27 肆、結果…………………………………………………………………29 一、尋找染色體中合適的基因插入區域………………………………29 二、在培養基中攜帶 avrPto 及 avrPtoB 基因的質體互補菌株其質體穩定性之測定………………………………………………29 三、於植物中攜帶 avrPto 及 avrPtoB 基因的質體互補菌株其質體穩定性之測定 …………………………………………………………30 四、確認 ΔavrPtoB 互補菌株………………………………………32 五、ΔavrPtoB插入染色體的互補菌株中,PSPTO_0286 及PSPTO_0287 表現情形…………………………………………………32 六、ΔavrPtoB插入染色體及以質體互補基因的互補菌株之基因表現情形.32 七、確認 Δhcp1、Δhcp2 和 Δhcp1/2 互補菌株…………………33 八、Δhcp1、Δhcp2 和 Δhcp1/2插入染色體的互補菌株中,PSPTO_0286 及PSPTO_0287 表現情形…………………………………33 九、Δhcp1、Δhcp2 和 Δhcp1/2插入染色體及以質體互補基因的互補菌株之基因表現情形…………………………………………………33 十、刪除 t6ss2 基因叢集 (cluster) 和 hcp2 基因使 Pst DC3000 的競爭力下降……………………………………………………………34 十一、以插入染色體方式所得之互補菌株可回復與野生株 Pst DC3000 相似程度的競爭力……………………………………………36 1.以質體互補 hcp1 基因會使 Δhcp1 於競爭試驗時的菌數下降..36 2.以插入染色體或以質體攜帶 hcp2 均能成功互補 Δhcp2 功能缺 陷…………………………………………………………………37 3.以插入染色體法在 Δhcp1/2 中互補hcp2的效果較以質體互補法佳…………………………………………………………………38 十二、野生型 E. coli strain MG1655 與 Pst DC3000 Δhcp1/2 突變株及互補菌株競爭試驗結果…………………………40 伍、討論………………………………………………………………41 一、Pst DC3000染色體中其他候選插入區域…………………………41 二、質體保留率的差異…………………………………………………42 三、插入基因序列數量…………………………………………………43 四、重組效率……………………………………………………………44 五、互補菌株的基因表現情形…………………………………………44 六、Pst DC3000的第六型分泌系統分泌作用蛋白不受溫度影響……44 七、第六型分泌系統參與 Pst DC3000 與其他細菌的競爭作用……45 八、Pst DC3000 競爭上為較弱勢的菌種……………………………46 九、hcp1質體互補菌株競爭力下降……………………………………47 十、hcp2染色體插入互補菌株互補效果較好…………………………48 陸、參考文獻……………………………………………………………50 圖表目錄 Table 1. Bacterial strains and plasmid used in this study60 Table 2. Primers used for plasmid construction………………62 Table 3. Primers used for semi-quantitagive RT-PCR…………63 圖一、Pst DC3000染色體中基因插入區域 (landing pad) 之附近基因位置圖,RT-PCR 分析 PSPTO_0286 及 PSPTO_0287 基因表現情形及 pK18msLP 載體構造圖………………………………………………64 圖二、培養基中各 Pst DC3000 菌株其質體 pCPP45 的穩定性測試65 圖三、番茄 (Moneymaker) 中質體保留率試驗結果…………………66 圖四、番茄 (RG-prf3) 中質體保留率試驗結果……………………67 圖五、番茄 (RG-pto11) 中質體保留率試驗結果……………………68 圖六、番茄 (RG-PtoR) 中質體保留率試驗結果……………………69 圖七、菸草 (Nicotiana benthamiana) 中質體保留率試驗結果…70 圖八、利用 PCR 確認插入染色體或質體互補基因方式之互補菌株中是否具有互補基因avrPtoB……………………………………………71 圖九、利用半定量 RT-PCR 分析以插入染色體或質體互補方式之互補菌株其 avrPtoB、PSPTO_0286 及 PSPTO_0287 表現情形基因表現情形…………………………………………………………………………72 圖十、利用西方墨點法分析以插入染色體或質體互補方式之互補菌株中,其AvrPtoB 蛋白質的表現及外泌情形……………………………73 圖十一、利用 PCR 確認插入染色體或質體互補基因方式之互補菌株中是否具有互補基因 hcp1或 hcp2……………………………………74 圖十二、利用半定量 RT-PCR 分析插入染色體互補菌株中 hcp1 互補菌株和hcp2 互補菌株的 PSPTO_0286 及 PSPTO_0287基因表現情形75 圖十三、利用半定量 RT-PCR 分析以插入染色體或質體互補方式之互補菌株其hcp1 及 hcp2 基因表現情形………………………………76 圖十四、利用西方墨點法分析以插入染色體或質體互補方式之互補菌株中,其 Hcp1 和 Hcp2 蛋白質的表現情形…………………………77 圖十五、利用西方墨點法分析 Δhcp2 突變株以插入染體或質體方式互補 hcp2基因後,其 Hcp2 蛋白質的累積及外泌情形……………78 圖十六、利用西方墨點法分析 Δhcp1/2 突變株以插入染體或質體方式互補 hcp2 基因後,其 Hcp2 蛋白質的累積及外泌情形…………79 圖十七、t6ss2 及 hcp2 與 Pst DC3000 對 E. coli 競爭能力有關…………………………………………………………………………80 圖十八、Pst DC3000 之 Δhcp1、Δhcp2 和 Δhcp1/2 突變株及互補菌株生長情形…………………………………………………………81 圖十九、hcp1 不參與 Pst DC3000 和 E. coli strain DH5α競爭作用上………………………………………………………………………82 圖二十、在與 E. coli strain DH5α 的競爭作用中,以插入染色體或質體互補均能恢復 Δhcp2 功能至野生株程度……………………83 圖二十一、Pst DC3000 Δhcp1/2 突變株以插入染色體或質體互補之互補菌株與 E. coli strain DH5α 競爭的結果……………………84 圖二十二、hcp1不參與 Pst DC3000 和 E. coli strain BLR (DE3) 競爭作用上………………………………………………………………85 圖二十三、在與 E. coli strain BLR (DE3) 的競爭作用中,以插入染色體或質體互補均能恢復 Δhcp2 功能至野生株程度……………86 圖二十四、Pst DC3000 Δhcp1/2 突變株以插入染色體或質體互補之互補菌株與 E. coli strain BLR (DE3) 競爭的結果………………87 圖二十五、hcp1不參與 Pst DC3000 和 E. coli strain BL21(DE3) 競爭作用上………………………………………………………………88 圖二十六、在與 E. coli strain BL21 (DE3) 的競爭作用中,以插入染色體或質體互補均能恢復 Δhcp2 功能至野生株程度…………89 圖二十七、Pst DC3000 Δhcp1/2 突變株以插入染色體或質體互補之互補菌株與 E. coli strain BL21(DE3) 競爭的結果………………90 圖二十八、Pst DC3000 Δhcp1/2 突變株以插入染色體或質體互補之互補菌株與 E. coli strain MG1655 競爭的結果…………………91 Figure A1. Schematic representation of a T6SS………………92 | |
dc.language.iso | zh-TW | |
dc.title | 於 Pseudomonas syringae pv. tomato DC3000 中染色體定點插入應用於基因表現之研究 | zh_TW |
dc.title | Development and application of a new site-specific chromosomal integration strategy for gene expression 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 | Pseudomonas syringae pv. tomato DC3000,互補,定點染色體插入,競爭試驗,質體喪失, | zh_TW |
dc.subject.keyword | Pseudomonas syringae pv. tomato DC3000,complementation,site-specific chromosomal integration,competition,plasmid loss, | en |
dc.relation.page | 92 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-08-15 | |
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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