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研究生:許雅文
研究生(外文):Ya-Wen Hsu
論文名稱:台灣蝴蝶蘭軟腐病原細菌Erwiniachrysathemi之特性及分子類型區分之研究
論文名稱(外文):Studies on characteristics and molecular typing of bacterial soft rot pathogen of Phalaenopsis, Erwinia chrysanthemi.
指導教授:李永安李永安引用關係
指導教授(外文):Yung-An Lee
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:179
中文關鍵詞:蝴蝶蘭軟腐病分子鑑定
外文關鍵詞:Erwinia chrysanthemiPhalaenopsis soft rotmolecular typing
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由台灣不同蝴蝶蘭栽培地區,分離蝴蝶蘭軟腐病病株之病原,分離出的蝴蝶蘭軟腐病菌皆均可在NGM培養基上呈藍色,indC PCR結果都可擴增出644 bp 大小的DNA 片段,證實台灣蝴蝶蘭的軟腐病均由Erwinia chrysanthemi引起。測試所分離E. chrysanthemi菌株之生理生化特性,菌株之特性接近biovar 3與biovar 4。測試9株軟腐病菌菌株 (依PFGE區分為不同分子類型)對藥劑之抗性,軟腐病菌菌株均可抗四種抗生素 (lincomycin 2 mg, novobiocin 5 mg, clindamycin 2 mg, vancomycin 30 mg)且對低量 (20 ppm)的銅和嘉賜黴素 (kasugamycin)也具有抗性,而有兩個菌株對streptomycin具有抗藥性,但所有菌株對tetracycline (6.25~25 ppm)及歐索林酸 (oxolinic acid) (6.25~25 ppm)均不具抗性。蝴蝶蘭軟腐病菌菌株對蝴蝶蘭不同品種,導致軟腐病病徵的能力也有差異,在測試的蝴蝶蘭不同品種中,均可被不同分子類型的菌株所感染,而當中蝴蝶蘭A03670品種較具抗耐病能力。再進一步將台灣蝴蝶蘭軟腐病菌與E. chrysanthrmi不同biovar之標準菌種做16S rRNA基因序列比對,結果兩者之間相似度為90~96%。台灣蝴蝶蘭軟腐病菌與biovar 3標準菌株的16S rRNA的序列相同度為90~92%而和biovar 4標準菌株的16S rRNA的序列相同度為94~96%。藉由pectate lyase ADE (pelADE)、recA、Ech 16S rRNA進行PCR-RFLP,台灣蝴蝶蘭軟腐病菌只有1種分子類型。在RFLP (restriction fragment length polymorphism)實驗中,藉由pectate lyase Z (pelZ)、indigoidine biosynthesis gene C (indC)、cellulase genes(cel5Z和cel8Y)等基因片段為探針,蝴蝶蘭軟腐病菌之菌株有1或2種分子類型。比較台灣蝴蝶蘭軟腐病菌與E. chrysanthemi不同biovar之標準菌種pectate lyase基因分布,pelADE、pelBC、pelI、pelW、pelX、pelZ基因當探針進行RFLP實驗,台灣蝴蝶蘭軟腐病菌的分子類型只有1或2種,而E. chrysanthemi不同biovar之標準菌種的分子類型1~8種。台灣軟腐病菌在pectate lyase的RFLP實驗結果與biovar 9較相似。由上述只針對某些基因的分析方法結果可知台灣蝴蝶蘭軟腐病菌菌株的變異性並不大,進一步再利用脈衝式電泳(pulsed field gel electrophoresis, PFGE)進行分子類型區分,卻發現台灣蝴蝶蘭軟腐病菌至少有13種分子類型。經由PFGE可發現菌株之間的較多差異,除發生點突變外,可能是菌株之間rRNA基因operon (rrn)間發生基因重組 (DNA rearrangement)或是基因組中發生DNA缺失 (deletion)或插入 (insertion)的情形。以I-CeuI (只作用在23S ribosomal RNA基因序列)作用台灣蝴蝶蘭軟腐病菌,再利用脈衝式電泳分析,發現台灣蝴蝶蘭軟腐病菌均可產生7條片段,表示菌株都具有7個rrn operon,但菌株間呈現13種PFGE 類型,顯示在基因組內發生DNA缺失 (deletion)及插入(insertion)。以I-CeuI進行部分酵素作用 (partial digestion),構築軟腐病菌菌株基因組的I-CeuI限制酵素圖譜,發現各菌株均具有多種可能性的組合排列,無法確定正確的組合情形。由Ech PB1 genomic library 選殖出Ech PB1的rrn operon並經由定序得到其左右基因,而建構Ech PB1限制圖譜。藉由設計rrn operon左右基因的primer對其他菌株進行PCR,PCR結果顯示台灣蝴蝶蘭軟腐病菌菌株並未在rrn operon中23S序列中發生homologous recombination,而E. chrysanthemi不同biovar之標準菌株,PCR的結果顯示在rrn operon中發生homologous recombination,台灣蝴蝶蘭軟腐病菌菌株間的基因組中rrn operon間未發生DNA重組情形,而導致台灣蝴蝶蘭軟腐病菌菌株間的差異,主要是在基因組中發生DNA缺失及插入的情形。台灣蝴蝶蘭軟腐病菌菌株Ech PB1,經由Ech PB1 genomic library 選殖出6個rrn operon,定序分析其左右基因,12個基因中有10個基因與Erwinia carotovora subsp. atroseptica SCRI1043相同,剩下2個基因(A12-B11和hdfR) 與Erwinia carotovora subsp. atroseptica SCRI1043基因不同,此結果顯示台灣軟腐病菌菌株Ech PB1的rrn operon之左右基因與Erwinia carotovora subsp. atroseptica SCRI1043相似。此外,Ech PB1與其他腸內科菌比較彼此左右基因共同的只有purH、murI、murB、hemG、yifA和mobB。
The causal agent of Phalaenopsis orchids soft rot was isolate from different orchid cultivation areas in Taiwan. All isolates grew well on the NGM medium and developed blue pigment, and contained indC as determined by PCR amplification. The causal agent of Phalaenopsis soft rot lesions was all identified as E. chrysanrhemi. Nine strains of Phalaenopsis soft rot pathogen tested chemical resistance did not show inhibition zone to four kinds of antibiotic disc. The soft rot pathogen had the resistance to low-level of copper and kasugamycine, and two strains had the resistance to streptomycin, but all nine strains had no resistance to tetracycline (6.25~25 ppm) and oxolinic acid (6.25~25 ppm). All isolates led to different symptom size on different sorts of Phalaenopsis. All isolates who molecular types were different infected all of the sorts of Phalaenopsis tested; Phalaenopsis A03670 had more disease resistant capacity. Sequence analysis of 16S rDNA gene of all isolates, revealed 90~96% of homology to 16S rDNA gene of type strains (the different biovars) of Erwinia. E. chrysanthemi from Taiwan Phalaenopsis soft rot and the type strain (biovar 3 and biovar 4) of Erwinia, revealed 90~92% and 94~96% , respectively of homology of 16S rRNA gene. The pelADE、recA and 16S rRNA fragments amplified from the E. chrysanthemi strains were compared by performing a PCR-RFLP analysis showed only one pattern. In RFLP experiment, pectate lyase Z (pelZ)、indigoidine biosynthesis gene C (indC) and cellulsase genes (cel5Z and cel8Y) were used as probe to perform Southern hybridization, it was found that E. chrysanthemi from Phalaenopsis soft rot has one or two molecular patterns. By using pectate lyase genes (pelADE、pelBC、pelI、pelW、pelX、pelZ) as probes in Southern hybridization to compare pectate lyase gene distribution of all isolates and different biovars of the type strains of E. chrysantehmi, the experiment found all isolates had only one or two different patterns, and between different biovars of the type strains of E. chrysantehmi were 1~8 different pattern. The pectate lyase distribution of all isolates was similar with biovar 8 of type strains of E. chrysantehmi. By using pulsed-field gel electrophoresis for molecular typing, it was founf that E. chrysanthemi from Taiwan Phalaenopsis soft rot contained at least 13 molecular types. Different molecular typing of E. chrysanthemi might be due to DNA rearrangement between rrn operon or DNA deletion and insertion. PFGE of I-CeuI enzyme digestion of E. chrysanthemi DNA produced seven I-CeuI fragments, which means the bacterium had seven rrn operons, but 13 different PFGE patterns were shown among strains. Partial digestion of E. chrysanthemi with I-CeuI showed many kinds possibility of I-CeuI fragments order. We construct the genomic library of Ech PB1, and obtained the sequence of rrn operon and its DNA flanking by sequencing. Then, restriction map were builded upon the sequence of rrn operon region. In PCR experiment, primers were designed base upon the sequence of DNA flanking each of the rrn operon in Ech PB1 to different strains, PCR results indicated that homologous recombination was most found in the rrn between E. chrysanthemi strains from Phalaenopsis soft rot, but occurred in different biovars of the type strains of E. chrysanthemi. Analysis DNA flanking of rrn operons within E. chrysanthemi and Erwinia carotovora subsp. atroseptica SCRI1043, the experiment found twelve genes of six rrn operons of Ech PB1 which had two genes (A12-B11 and hdfR) which is different from Erwinia carotovora subsp. atroseptica SCRI1043. Ech PB1 with homologous flanking genes had purH、murI、murB、hemG 、yifAand mobB which were the same as other bacterium. DNA rearrangement was not found in rrn operon between E. chrysanthemi strains from Taiwan Phalaenopsis soft rot, and DNA deletion and insertion of genome leads to different molecular types of E. chrysanthemi in Taiwan.
目 錄
蝴蝶蘭軟腐病Erwinia chrysanthemi之特性及分子多樣性探討
中文摘要
英文摘要
前言
材料與方法
菌種的取得、培養條件及儲存的方法
以NGM培養基自蝴蝶蘭軟腐病株分離軟腐病菌
軟腐病菌indC基因片段之PCR反應
軟腐病菌菌株 biovar鑑定之生理生化測試
碳源測試
NaCl的耐受性測試
Gelatin liquefaction
Arginine dehydrolase
39 ℃生長測試
軟腐病菌菌株對藥劑之抗性
抗生素紙錠
測試銅離子
農業抗生素
軟腐病菌菌株對蝴蝶蘭品種的致病能力
蝴蝶蘭的品種及植株大小
細菌懸浮液濃度及配製方法
蝴蝶蘭軟腐病菌的接種實驗
軟腐病菌菌株之16S rRNA基因選殖、定序及比對
NJ(neighborjoining method)劃親緣關係的步驟
軟腐病菌菌株的分子類型(molecular typing)
PCRRFLP
RFLP
果膠質裂解酶(pectate lyase)基因種類分布
Pulsedfield gel electrophoresis
E. chysanthemi rRNA基因operon(rrn)間的基因重組(DNA rearrangement)
ICeuI Pulsedfield gel electrophoresis(PFGE)
建構軟腐病菌Ech PB1之ICeuI DNA片段之限制酵素圖譜(restriction map)
以ICeuI partial digestion建構限制圖譜
ICeuIPFGE DNA片段含rrn operon兩旁基因種類的建構限制圖譜
自Ech PB1基因庫(genomic library)選殖rrn operons
rrn operon兩旁基因核苷酸定序及分析
確定ICeuIPFGE DNA片段含rrn operon兩旁基因種類
軟腐病菌菌株rrn operon 間的基因重組
軟腐病菌及其他細菌rrn operon兩旁基因種類之分析
基本分生實驗
實驗結果
以NGM培養基自蝴蝶蘭軟腐病株分離軟腐病菌
軟腐菌 biovar鑑定之生理生化測試
軟腐病菌菌株對藥劑之抗性
軟腐病菌菌株對蝴蝶蘭品種的致病能力
16S rRNA基因選殖、定序及比對
軟腐病菌菌株之分子鑑定(molecular typing)
PCRRFLP
RFLP
果膠質裂解酶pectate lyase基因種類分布
Pulsedfield gel electrophoresis
E. chysanthemi rRNA基因operon(rrn)間的基因重組(DNA rearrangement)
ICeuI Pulsedfield gel electrophoresis
建構軟腐病菌Ech PB1之ICeuI之限制酵素圖譜
以ICeuI partial digestion建構限制圖譜
ICeuIPFGE DNA片段含rrn operon兩旁基因種類的建構限制圖譜
軟腐病菌菌株rrn operon間的基因重組
軟腐病菌及其他細菌rrn operon兩旁基因種類分析
討論
參考文獻
圖表
附錄
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