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研究生:廖惠玲
研究生(外文):Huei-Ling Liao
論文名稱:台灣植物癌腫病菌之PCR鑑定及檢測
論文名稱(外文):PCR-Based Identification and Detection of Agrobacterium tumefaciens in Taiwan
指導教授:陳昭瑩陳昭瑩引用關係
指導教授(外文):Chao-Ying Chen
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
中文關鍵詞:聚合酵素連鎖反應Ti 質體毒性基因癌腫病玫瑰紫花宿苑
外文關鍵詞:PCRAgrobacterium tumefaciensTi plasmidpolymerase chain reactionT-DNArose (Rosa hybrida)aster (Aster ericoides L)crown gall disease
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Agrobacterium tumefaciens (Smith and Townsend) Conn為植物細菌性癌腫病菌,會在多數雙子葉植物及某些單子葉植物的傷口上侵入感染,刺激植物細胞不斷地增生,造成腫瘤病徵,被感染的植物會呈現發育不良,無法正常生長的病症。聚合酵素連鎖反應(polymerase chain reaction, PCR)是一簡單、敏感且快速的偵測技術,即使目標細菌數量相當少時,也能偵測得到。在本研究中,依據A. tumefaciens Ti質體基因- virA、virD1、virC1-D2、acs、nos、ocs、6a及iaaH所設計的八對引子,在玫瑰及紫花宿苑癌腫病菌擴增的PCR產物圖譜並不相同,各有五對引子可分別自玫瑰及紫花宿苑癌腫病菌獲得偵測訊號,這八對引子均無法在10種其他屬的細菌獲得偵測訊號。另外,virC1-D2引子對的PCR-RFLP結果也顯示台灣玫瑰及紫花宿苑癌腫病菌的Ti質體歸屬於不同類型。進一步利用可直接自玫瑰癌腫病菌細胞中擴增預期DNA片段的三對引子(virC1-D2、iaaH及acs),以人工接種的迷你玫瑰植株建立癌腫病菌的檢測流程,包括細菌增量培養及PCR鑑定。結果顯示在盆栽迷你玫瑰及田間玫瑰罹病株的無腫瘤病徵莖部組織中均有癌腫病菌的存在,癌腫病菌可移動至腫瘤部位上方或下方莖部組織及與腫瘤相距甚遠的側枝及幼嫩枝條,具有系統性分佈的特性。另一方面,細菌生理生化特性分析結果顯示台灣玫瑰及紫花宿苑癌腫病菌與癌腫病菌生物型I及生物型II有明顯的差異。但根據16S rDNA的PCR-RFLP圖譜比對仍可將台灣玫瑰及紫花宿苑癌腫病菌歸屬於生物型I。
Agrobacterium tumefaciens (Smith and Townsend) Conn is a phytopathogenic bacterium that causes crown gall disease. A. tumefaciens generally infects the wounded tissues in most dicotyledonous and some monocotyledonous plants to incite tumorous growth of plant cells and leads to the formation of gall tissues. The growth of diseased plants in the nursery are generally retarded.
Polymerase chain reaction (PCR) is a simple, sensitive, and rapid method to detect microorganisms in a low amount. In this study, eight pairs of oligonucleotide primers based on the sequences of virA、virD1、virC1-D2、acs、nos、ocs、6a and iaaH on Ti plasmid of A. tumefaciens were used to differentiate rose and aster strains of A. tumefaciens in Taiwan. Different sets of primer pairs (each including five primer pairs) could amplify the target DNAs from tumorigenic rose or aster strain. None of the tested primer pairs could detect the ten kinds of bacteria in other genus. In addition, the PCR-RFLP analysis with primer pair virC1-D2 showed that the Ti plasmids in the rose and aster strains were in different groups.
With three primer pairs (virC1-D2, iaaH, and acs) amplifying the target DNAs from boiled agrobacterial cells, an examination and detection procedure for the pathogenic agrobacteria in the rose plants was established in a combination of bacterial enrichment and PCR identification. The existence of A. tumefaciens in the symptomless stems of pot and field rose plants was verified. The results showed that pathogenic agrobacteria could migrate upward or downward to the sites of rose stems very far away from the gall tissues, indicating that A. tumefaciens could systemically distribute in the rose plants.
In addition, the analysis of physiological and biochemical characteristics showed that the patterns of rose and aster strains of A. tumefaciens in Taiwan were distinct from that of biovar I and biovar II. However, the rose and aster strains of A. tumefaciens in Taiwan could be categorized as biovar I according to the PCR-RFLP patterns of 16S rDNA.
壹、 中文摘要……………………………………………………………………..1
貳、 英文摘要……………………………………………………………………..2
參、 前言…………………………………………………………………………..4
肆、 前人研究……………………………………………………………………..6
伍、 材料與方法………………………………………………………………….16
陸、結果
一、台灣玫瑰及紫花宿苑癌腫病菌特性………………………………… ….35
1. 病原性測定………………………………………………………… …35
2. 革蘭氏染色…………………………………………………… ………35
3. 生理生化特性…………………………………………………… ……36
4. Biolog分析………………………………………………………… ….37
5. 16S rDNA之PCR-RFLP圖譜分析…………………………… ……. 37
二、Agrobacterium對照菌株之PCR分析…………………………… …..37
1. 植物癌腫病原菌染色體干擾virA引子對之PCR擴增反應分析... ..38
2. 以vir基因為偵測目標之PCR分析…………………….…...….….. .38
3. 以T-DNA序列為偵測目標之PCR分析…………………….… …...39
三、台灣玫瑰及紫花宿苑癌腫病菌之PCR分析…………… ………..….39
1. Ti質體之檢視………………………………………………… ……....39
2. 以vir基因為偵測目標之PCR分析……………………………… ...40
3. 以T-DNA序列為偵測目標之PCR分析………………………… ...40
四、virC1-D2及virA引子對偵測癌腫病菌敏感度分析…………… …...40
五、建立玫瑰植株中癌腫病菌之檢測系統……………………… …….40
1. 罹患癌腫病玫瑰無病徵莖部組織汁液之PCR檢測……………… .…...41
2. 罹患癌腫病玫瑰正常莖部組織癌腫病菌分離菌株之PCR分析…...… .41
3. 罹患癌腫病玫瑰莖部組織正常莖部組織非癌腫病菌
分離菌株之PCR分析……………………………...………………… …42
4. 罹患癌腫病玫瑰正常根部組織汁液之PCR檢測………………….… ...42
5. 健康玫瑰植株莖部組織汁液之PCR檢測……………………………... .42
六、玫瑰莖部組織汁液干擾PCR反應之分析……………………………..…43
七、玫瑰病株無病徵莖部組織中所含癌腫病菌數量之估計…………… ...43
八、感染玫瑰植株癌腫病菌之最低菌量…………………………… ……....43
九、台灣玫瑰及紫花宿苑癌腫病菌virD1基因選殖及序列分析…… ……...44
十、台灣玫瑰及紫花宿苑癌腫病菌virC1-D1-D2 DNA之
PCR-RFLP圖譜……………………………………………………….. .44
柒、討論
一、 台灣玫瑰及紫花宿苑癌腫病菌特性………………………………… ...46
二、 對照菌株之PCR分析……………………………………………… …..46
三、 台灣玫瑰及紫花宿苑癌腫病菌之PCR分析…………………………. .49
四、 台灣玫瑰及紫花宿苑癌腫病菌virD1基因序列分析……………… ….50
五、 台灣玫瑰及紫花宿苑癌腫病菌virD1基因之PCR-RFLP分析…… ….51
六、 在玫瑰植株上建立癌腫病菌之檢測系統……………………………… .51
七、 台灣玫瑰癌腫病菌於玫瑰植株之系統性分佈………………………… .52
捌、 參考文獻
玖、 圖表集
拾、附錄
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