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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉瑞芬 | |
| dc.contributor.author | Ming-Wei Lai | en |
| dc.contributor.author | 賴名威 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:55:13Z | - |
| dc.date.available | 2023-08-02 | |
| dc.date.copyright | 2018-08-02 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-07-28 | |
| dc.identifier.citation | Ah-Fong, A.M.V., Bormann-Chung, C.A., and Judelson, H.S. 2008. Optimization of transgene-mediated silencing in Phytophthora infestans and its association with small-interfering RNAs. Fungal Genet. Biol. 45: 1197–1205.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71142 | - |
| dc.description.abstract | 病原菌侵染植物寄主時，會分泌各種細胞壁分解酵素以促進侵染進程，其中包含木聚醣酶 (endo-beta-1,4-xylanase)，能夠水解半纖維素的主要成分－長鏈木聚醣，達到破壞植物細胞壁的目的。本研究探討木聚醣酶在疫病菌(Phytophthora parasitica)致病過程中所扮演的角色，將疫病菌木聚醣酶基因家族中共四個基因進行選殖，並將之個別命名為ppxyn1、ppxyn2、ppxyn3與ppxyn4。胺基酸序列分析顯示這四個疫病菌木聚醣酶皆具有木聚醣酶的保守性酵素活性區，親緣關係分析顯示，有別於真菌木聚醣酶，疫病菌木聚醣酶全都隸屬於醣苷水解酶家族10 (glycoside hydrolases family 10, GH10)的成員。以嗜甲醇酵母菌(Pichia pastoris)表現重組蛋白進行測試，證實ppxyn1、ppxyn2及ppxyn4重組蛋白皆具有水解樺木木聚醣之酵素活性，並且在耐熱特性及所需最適反應條件(包括酸鹼性及作用溫度等)皆有不同。以定量反轉錄-聚合酶連鎖反應測定基因表現情形，發現ppxyn1及ppxyn2在疫病菌感染番茄葉片初期即大量表現，ppxyn3在靜止子(cyst)表現量較高，而ppxyn4則在萌發靜止子被誘導表現。為進一步了解疫病菌木聚醣酶在疫病菌致病過程的的重要性，本研究應用Gateway選殖系統改造靜默載體，並建立疫病菌轉型系統，可以雙股 RNA 驅動疫病菌之基因靜默。分析結果顯示ppxyn1及ppxyn2靜默轉型株在圓葉煙草(Nicotiana benthamiana)或番茄(Solanum lycopersicum)上所造成的病徵皆有減弱現象，顯示ppxyn1及ppxyn2是疫病菌侵染植物時重要的細胞壁分解酵素。此外，利用農桿菌注入法所進行的分析發現ppxyn1及ppxyn3在圓葉煙草造成壞死斑，ppxyn2則在煙草(Nicotiana tabacum cv. Xanthi)上造成壞死斑，顯示這些基因可能具有導致細胞壞死的活性。 | zh_TW |
| dc.description.abstract | To infect their host plants, pathogens need to secret a variety of cell wall degrading enzymes, including endo-beta-1,4-xylanase that is crucial for the initial breakdown of xylan, the major hemicellulose component of the plant cell wall. To investigate the role of xylanase in Phytophthora parasitica, an oomyceteous plant pathogen with a wide host range, four genes encoding endo-b-1,4-xylanase, which named ppxyn1, ppxyn2, ppxyn3, and ppxyn4, were cloned and characterized respectively. Analysis of the deduced amino acid sequences indicated that all these genes contain active site signature for xylanase and belong to glycoside hydrolases family 10 (GH10). Phylogenic analysis demonstrated that all these genes form a cluster distinct from xylanases of the fungal pathogens. Analysis of the recombinant proteins obtained from the yeast Pichia pastoris indicated that ppxyn1, ppxyn2, and ppxyn4 encode a functional protein with a hydrolase activity toward birchwood xylan. However, each protein behaved differently regarding to their thermostability and conditions for optimal enzymatic activity. Analysis by quantitative reverse transcriptase-PCR indicated that ppxyn1 and ppxyn2 were up-regulated upon infection of tomato leaves by P. parasitica, suggestive of their involvement in the process of host infection. In contrast, ppxyn3 was highly expressed in cysts and ppxyn4 in germinating cysts. To further investigate the role of ppxyn genes in P. parasitica, silencing vectors were modified to facilitate Gateway cloning and CaCl2¬¬-PEG-mediated protoplast transformation method was established, which allow dsRNA mediated gene silencing in this pathogen. Both ppxyn1- and ppxyn2-silenced transformants show reduced virulence on tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum) plants. These results demonstrate the crucial role of xylanase-encoding ppxyn1 and ppxyn2 in the infection process of P. parasitica. In addition, transient expression of ppxyn1 and ppxyn3 by agroinfiltration caused necrosis on the leaves of N. benthamiana. As well, ppxyn2 caused necrosis on the leaves of Nicotiana tabacum cv. Xanthi. These results suggest that these genes might have necrotizing activities. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:55:13Z (GMT). No. of bitstreams: 1 ntu-107-F95633016-1.pdf: 53138079 bytes, checksum: 92732a8bb8af394d571811bd176337e1 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員會審定書......i
摘要......ii Abstract......iii Contents......v Chapter I Introduction......1 I-1 Xylanase (Beta-1,4-D-endoxylanase; EC 3.2.1.8)......1 I-2 The roles of xylanase in pathogenicity of plant fungal pathogens......2 I-3 The role of CWDEs in plant immunity......3 I-4 The role of xylanase in plant immunity......5 I-5 Phytophthora parasitica and establishment of methods for its transformation......6 I-6 Gateway cloning system......9 I-7 The aims of this study......10 Chapter II Materials and Methods......11 II-1 Phytophthora parasitica culture and inoculation......11 II-2 Growth condition of plant materials......12 II-3 Molecular cloning and sequence analysis of genes encoding xylanase in Phytophthora parasitica......12 II-4 Southern blot hybridization......13 II-5 Expression of recombinant protein in Pichia pastoris......13 II-6 Expression of recombinant protein in Escherichia coli......14 II-7 Assay of the xylanase activity......15 II-8 Isolation of total RNAs......16 II-9 Quantitative reverse transcriptase-PCR (qRT-PCR)......16 II-10 Construction of the Silencing Vector......17 II-11 Construction of vectors for dsRNA-mediated gene silencing......18 II-12 Polyethylene glycerol (PEG)-mediated transformation of Phytophthora parasitica......18 II-13 Pathogenicity test......20 II-14 PVX Agro-infection Assay......21 II-15 Protein transient expression by agroinfiltration......21 II-16 Isolation of apoplastic fluid......22 Chapter III Establishment of a method for dsRNA mediated gene silencing in Phytophthora parasitica......23 III-1 Establishment of methods for gene overexpression in Phytophthora parasitica......23 III-2 Establishment of methods for double-stranded RNA (dsRNA)-mediated gene silencing in Phytophthora parasitica......24 III-3 Discussion......26 Chapter IV ppxyn1 and ppxyn2 play important roles in pathogenesis of Phytophthora parasitica......31 IV-1 Molecular cloning and sequence analysis of a xylanase gene family from Phytophthora parasitica......31 IV-2 Assay for the enzymatic activity of ppxyn recombinant proteins......33 IV-3 Expression pattern of ppxyn in different life stages of Phytophthora parasitica......35 IV-4 The role of ppxyn in the pathogenicity of Phytophthora parasitica......36 IV-5 Discussion......39 Chapter V Investigation of the roles of ppxyns in plant immunity......44 V-1 Necrotizing activity assay for the ppxyn genes by transient protein expression......44 V-2 Discussion......45 Reference......49 Tables......66 Table 1. List of oligonucleotide primers used in this study......66 Table 2. Characteristics of proteins encoded by the xylanase-encoding genes of Phytophthora parasitica......68 Table 3. Amino acid composition of each PPXYN......69 Figures and Legends......70 Figure 1. Map of the vectors for double-stranded RNA mediated gene silencing in Phytophthora parasitica.......71 Figure 2. Expression of green fluoresces protein by Phytophthora parasitica transformants carrying pEX::eGFP.......73 Figure 3. Reverse transcriptase-PCR to amplify partial sequences of putative xylanase-encoding genes.......75 Figure 4. Screening of the genomic library of Phytophthora parasitica for putative xylanase-encoding genes.......76 Figure 5. Multiple sequence alignment of nucleotide sequences from ppxyn1, ppxyn2, ppxyn3, and ppxyn4.......77 Figure 6. Genomic Southern blot analysis of Phytophthora parasitica.......78 Figure 7. Multiple sequence alignment of deduced amino acid sequences from ppxyn1, ppxyn2, ppxyn3, and ppxyn4.......79 Figure 8. Phylogenetic analysis of the xylanase genes.......80 Figure 9. The electrophoretic pattern of ppxyn recombinant proteins.......81 Figure 10. Enzymatic activity assays of ppxyn recombinant proteins.......82 Figure 11. Analysis of the enzymatic activity of ppxyn3 and ppxyn4 recombinant proteins obtained from Pichia pastoris by using different substrates.......84 Figure 12. The electrophoretic pattern of ppxyn recombinant proteins obtained from Escherichia coli.......86 Figure 13. Thermostability and durability of ppxyn recombinant proteins.......87 Figure 14. Quantification of the transcripts of xylanase genes by real-time quantitative reverse transcriptase-PCR.......88 Figure 15. PCR analysis of putative ppxyn1- and ppxyn2-silenced transformants of Phytophthora parasitica.......90 Figure 16. The morphology of sporangia produced by ppxyn1- and ppxyn2-silenced transformants of Phytophthora parasitica.......91 Figure 17. Downregulation of ppxyn1 and ppxyn2 reduced virulence in Phytophthora parasitica.......93 Figure 18. ppxyn1- and ppxyn2-silenced transformants showed reduced virulence toward Nicotiana benthamiana.......95 Figure 19. Systemic expression of ppxyn genes on Nicotiana benthamiana by agroinfection.......97 Figure 20. Systemic expression of ppxyn genes on Solanum lycopersicum by agroinfection.......99 Figure 21. Transient expression of ppxyn on Nicotiana benthamiana by agroinfiltration.......101 Figure 22. Transient expression of ppxyn on Nicotiana tabacum and Solanum lycopersicum by agroinfiltration.......103 Supplementary figure 1. Map of the plasmid construct for recombinant protein expression in Pichia pastoris.......105 Supplementary figure 2. Map of the plasmid construct for recombinant protein expression in Escherichia coli.......106 Supplementary figure 3. Construction of binary vector for protein expression by agroinfection.......107 Supplementary figure 4. Construction of binary vector for protein expression by agroinfiltration.......108 | |
| dc.language.iso | en | |
| dc.subject | 木聚醣? | zh_TW |
| dc.subject | 疫病菌 | zh_TW |
| dc.subject | 致病性 | zh_TW |
| dc.subject | 細胞壁分解酵素 | zh_TW |
| dc.subject | 壞死 | zh_TW |
| dc.subject | cell wall degrading enzyme | en |
| dc.subject | Phytophthora parasitica | en |
| dc.subject | virulence | en |
| dc.subject | necrosis | en |
| dc.subject | xylanase | en |
| dc.title | 疫病菌木聚醣酶基因的選殖與功能性分析 | zh_TW |
| dc.title | Cloning and functional characterization of genes encoding xylanase in Phytophthora parasitica | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 曾顯雄,沈偉強,李敏惠,林乃君 | |
| dc.subject.keyword | 木聚醣?,疫病菌,致病性,細胞壁分解酵素,壞死, | zh_TW |
| dc.subject.keyword | xylanase,Phytophthora parasitica,virulence,cell wall degrading enzyme,necrosis, | en |
| dc.relation.page | 108 | |
| dc.identifier.doi | 10.6342/NTU201802052 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-07-30 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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