Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/95777
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dc.contributor曾國欽zh_TW
dc.contributor.author張世杰zh_TW
dc.contributor.authorShih-Chieh Changen_US
dc.contributor.other植物病理學系所zh_TW
dc.date2017zh_TW
dc.date.accessioned2018-10-12T04:01:32Z-
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Characterization of phenotypically distinct strains of Xanthomonas axonopodis pv. citri from Southwest Asia. Eur. J. Plant Pathol. 104, 477-487.zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/95777-
dc.description.abstract柑橘潰瘍病 (citrus canker) 為全球柑橘產業之重要病害,其病原菌 Xanthomonas citri subsp. citri (簡稱 Xcc) 為革蘭氏陰性、好氣之桿狀細菌,可感染芸香科各柑橘品種;果膠分解酵素可分解植物細胞壁,造成植物組織軟化、細胞質滲漏,並導致細胞死亡,為植物病原細菌之重要致病因子。本研究利用 β-glucuronidase (GUS) 做為報導蛋白,研究 Xcc strain XW19果膠分解酵素基因 pel1、pel2、pel3、pglA 及 peh-1之啟動子表現能力與其調控因子,並以基因缺失法針對 pel1、pel3、pglA 及 peh-1 等基因進行突變,探討其功能及對 Xcc strain XW19 致病過程的影響,發現 pel1、pel3、pglA 及 peh-1 基因啟動子之表現受 sodium polypectate、XVM2 培養基及柑橘葉片誘導且表現能力與細菌族群生長呈正相關,顯示 pel1、pel3、pglA 及 peh-1 可能與 Xcc strain XW19 之致病能力有關,然而只有 pel1 基因之表現受葡萄糖引起之分解代謝物抑制作用所抑制;pel3 基因啟動子於所有測試條件下皆可表現出最高之 GUS 活性,而 pel2 基因啟動子不論於培養條件下或植物體內皆測不到其表現量。接種突變菌株 XW19 ΔpglA 及 XW19 Δpeh-1 於柑橘葉片中,其病斑形態與病徵發展情形皆不受影響,推測 pglA 與 peh-1 基因可能與潰瘍病徵之發生無關,其功能尚待驗證;pel1 基因缺失會降低果膠裂解酵素活性,但其突變菌株 XW19 Δpel1並未造成柑橘葉片上病斑形態與病徵發展情形之變化,顯示 pel1 基因雖具分解果膠質的能力,但非影響病斑水浸狀邊緣之唯一因子;接種突變菌株 XW19 Δpel3 於柑橘葉片中所產生之病斑較小且發病時間較晚,顯示Pel3 為 Xcc strain XW19 致病過程中之毒力因子,影響潰瘍病病徵之誘發及細菌在寄主植物體中族群生長情形。此外,為探討限制引起非典型病徵之 Xcc strain XW121 於寄主植物內生長的因素,利用 PacBio RSII sequencing platform 進行基因體解序,獲得 4 個重疊群,序列資料以 Magnifying Genome (MaGe) Annotation Platform 分析發現 Xcc strain XW121 具有 1 個大小為 5,194,029 bp 之染色體及 2 個大小分別為 88,415 bp 與 16,610 bp 之質體,基因體全長為 5,299,054 bp,G+C content 為 64.7%,基因體含 5,168 個 putative CDS,具 2 套 rRNA operons、56 個 tRNA 及 6 個 misc_RNA,其中 3,539 個 CDS 可被歸類於 COG functional classes,佔基因體之 68.48%,尚有 1,629 個 CDS 無法被定義。利用 Roary pipeline 及 SiLiX software 分析顯示 Xcc strain XW121 在演化上屬於 A 病原型且較接近分離自中國大陸之柑橘潰瘍病菌株,與 A、Aw 及 A* 病原型進行比對分析後發現,Xcc strain XW121 具有 368 個 CDS 為其菌株所獨有,其中 334 個為未知功能基因、3 個為 transposase 及 31 個具預測功能之基因,其重要性尚待了解;此外,Xcc strain XW121 相較於 Xcc strain 306 缺少 XynB2、PthA1、PthA2、XopC2、XopAK 及 Hpa1 等致病因子,這些基因與非典型病徵之產生及細菌族群生長受限之關聯性尚待進一步實驗證實。zh_TW
dc.description.abstractCitrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive diseases of citrus. The pectolytic enzymes produced by phytobacteria are important virulence factors involved in tissue maceration, electrolyte loss and cell death of host plants. In this study, the promoter activity of the pectolytic enzyme genes pel1, pel2, pel3, pglA, and peh-1 were investigated in Xcc strain XW19 using the β-glucuronidase (GUS) gene as a reporter, and the function of pel1, pel3, pglA, and peh-1 were investigated using the gene deletion mutagenesis. GUS activity expressed under the control of the pel1, pel3, pglA, and peh-1 gene promoters positively correlated with bacterial growth, and displayed high GUS activity in the presence of sodium polypectate and induced in XVM2 medium and host plant. However, only pel1 was subjected to catabolite repression by glucose. GUS activity was significantly enhanced in the XW19-derived reporter strains after they were inoculated into the leaves of Mexican lime and grapefruit, suggesting the involvement of the pel1, pel3, pglA, and peh-1 genes in XW19 pathogenesis. The pel3 promoter produced the highest GUS activity under all test conditions, whereas no GUS activity was detected using the pel2 promoter in vitro and in planta. In comparison with wild type XW19, the canker lesions elicited by the pel1, pglA or peh-1 mutant was the same on the leaves of Mexican lime and grapefruit. Although, the pel3 mutant displayed reduced growth and induced smaller canker lesions on the leaves of Mexican lime or grapefruit compared with the wild type XW19, we demonstrate that Pel3 of Xcc strain XW19 is a virulence factor, and affects the canker elicitation and bacterial growth in host plant. In addition, the PacBio RSII sequencing platform was used to obtain a complete sequence of Xcc strain XW121, an Ar pathogenic variant in Taiwan. Based on Magnifying Genome (MaGe) Annotation Platform analysis, Xcc strain XW121 has a single, circular chromosome and 2 plasmids. The genome size is 5,299,054 bp and the G+C content averages 64.7%. The Xcc strain XW121 genome contains 5,168 putative CDSs, 2 rRNA operons, 56 tRNAs, and 6 misc_RNAs. There were 3,539 CDSs (68.48%) could be assigned to one or more COG functional classes, whereas there was not enough evidence for 1,629 CDSs to be assigned to any COG category. Phylogenetic analysis revealed that Xcc strain XW121 was grouped into the same cluster with A pathotype and more closely related with strains isolated from China. We also compared Xcc strain XW121 to the genome of Xcc strains 306, NCPPB 3608, and JJ238-24. Comparative genomic analysis showed 368 CDSs are unique to XW121, of which 334 are hypothetical genes, 3 are transposase, and 31 are singletons with predicted functions. In addition, XynB2, PthA1, PthA2, XopC2, XopAK, and Hpa1 were absent from Xcc strain XW121. These virulence factors might be responsible for the atypical symptom-producing and bacterial growth in host plant.en_US
dc.description.tableofcontents中文摘要…………………………………………………………………i Abstract..……………………………………………………………iii 目次………………………………………………………………v 表目次………………………………………………………………viii 圖目次……………………………………………………………………ix 第一章、緒言……………………………………………………………1 第二章、柑橘潰瘍病菌果膠分解酵素表現之影響因子及功能之探討 摘要………………………………………………………………………27 前言………………………………………………………………………28 材料方法 供試菌株與培養基……………………………………………………30 染色體 DNA 之萃取與濃度之測定…………………………………31 Escherichia coli熱休克法勝任細胞製備及熱休克法………………31 質體 DNA 之抽取與限制酵素之分析………………………………32 選殖片段之解序與比對………………………………………………33 X. citri subsp. citri strain XW19及其衍生菌株電轉型勝任細胞之製備……………………………………………………………………34 X. citri subsp. citri strain XW19 果膠分解酵素基因啟動子片段之選殖……………………………………………………………………34 果膠分解酵素基因啟動子表現載體之構築…………………………35 果膠分解酵素基因缺失突變之構築 (1) pel3 與 peh-1 基因缺失突變之構築…………………………35 (2) pel1 與 pglA 基因缺失突變之構築…………………………37 南方雜合法 (Southern hybridization)…………………………38 果膠分解酵素基因啟動子於不同培養條件下之表現………………40 果膠分解酵素基因啟動子於植物體上之表現………………………40 β-glucuronidase 活性測試 ─ 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside (X-Gluc) 組織染色法………………………41 β-glucuronidase 活性測試 ─ 4-methylumbelliferone (4-MU) 螢光測定法………………………………………………………………41 利用Thiobarbituric acid (TBA) 進行果膠分解酵素之活性測定........ 42 病原性測試…………………………………………………………….43 X. citri subsp. citri strain XW19 衍生菌株於墨西哥萊姆葉片之族群變化………………………………………………………………43 統計分析………………………………………………………………44 結果 果膠分解酵素基因啟動子片段之選殖………………………………44 pel1、pel3、pglA 及 peh-1基因於無菌培養下之表現具差異性…44 果膠質對果膠分解酵素基因之表現具不同之調控能力……………45 pel1、pel3、pglA 及 peh-1基因受 XVM2 培養基誘導表現……46 pel1、pel3、pglA 及 peh-1基因受柑橘葉片誘導表現…………46 果膠分解酵素基因缺失突變菌株構築之偵測 (1) pel3 基因缺失突變之偵測……………………………………47 (2) peh-1 基因缺失突變之偵測…………………………………48 (3) pel1 基因缺失突變之偵測……………………………………48 (4) pglA 基因缺失突變之偵測……………………………………48 pel1 基因缺失會降低 X. citri subsp. citri strains XW19 之果膠裂解酵素活性………………………………………………………49 pel3 基因影響 X. citri subsp. citri strains XW19 於寄主植物葉片中之病勢發展與族群生長………………………………………49 討論………………………………………………………………………50 參考文獻…………………………………………………………………55 Abstract..……………………………………………………………62 圖表………………………………………………………………………63 第三章、引起非典型潰瘍病徵之柑橘潰瘍病菌菌株XW121之基因體分析 摘要…………………………82 前言………………………………………………………………………83 材料方法 供試菌株與培養基……………………………………………85 染色體 DNA 之萃取與濃度之測定………………………………86 基因體定序與組裝…………………………………………………87 基因體註解與對比分析……………………………………………….87 親緣性分析……………………………………………………………88 結果 基因體解序結果……………………………………………………….88 基因體組成分析……………………………………………………….88 親緣性分析…………………………………………………………….89 X. citri subsp. citri strain XW121 與 Xcc strain 306 之基因體比對分析……………………………………………………………………89 第二、三、四型分泌系統與致病因子分析…………………………90 討論………………………………………………………………………91 參考文獻………………………………………………………………99 Abstract...…………………………………………………………108 圖表……………………………………………………………………109 附錄…………………………………………………………………126zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2020-02-06起公開。zh_TW
dc.subject柑橘潰瘍病菌zh_TW
dc.subject果膠分解酵素zh_TW
dc.subject啟動子活性zh_TW
dc.subject基因缺失zh_TW
dc.subject非典型病徵zh_TW
dc.subject基因體解序zh_TW
dc.subject致病因子zh_TW
dc.subjectXanthomonas citri subsp. citrien_US
dc.subjectpectolytic enzymeen_US
dc.subjectpromoter acitivityen_US
dc.subjectgene deletionen_US
dc.subjectpectate lyase activityen_US
dc.subjectatypical symptomen_US
dc.subjectwhole genome sequencingen_US
dc.subjectvirulence factoren_US
dc.title台灣柑橘潰瘍病菌果膠分解酵素基因之表現與功能分析暨基因體分析zh_TW
dc.titleExpression and functional analysis of pectolytic enzyme genes and genome analysis of Xanthomonas citri subsp. citri Taiwan strainsen_US
dc.typethesis and dissertationen_US
dc.date.paperformatopenaccess2020-02-06zh_TW
dc.date.openaccess2020-02-06-
item.cerifentitytypePublications-
item.grantfulltextrestricted-
item.languageiso639-1zh_TW-
item.fulltextwith fulltext-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypethesis and dissertation-
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