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研究生:林貝珊
研究生(外文):Bey-Shan Lin
論文名稱:火鶴花細菌性葉枯病病原菌核酸探針及PCR引子之研發與應用
論文名稱(外文):Development and application of cloned DNA probes and PCR primers for Xanthomonas campestris pv. dieffenbachiae, the causal agent of anthurium blight
指導教授:林長平林長平引用關係
指導教授(外文):Chan-Pin Lin
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:1999
畢業學年度:87
語文別:中文
論文頁數:86
中文關鍵詞:火鶴花細菌性葉枯病火鶴花細菌性葉枯病病原菌核酸探針聚合酵素連鎖反應引子對差別性雜配反應
外文關鍵詞:anthurium blightXanthomonas campestris pv. dieffenbachiaeDNA probepolymerase chain reaction (PCR)primer pairdifferential hybridization
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在本論文中,成功地研發出兩對可應用於以聚合酵素連鎖反應 (polymerase chain reaction,簡稱PCR) 技術偵測火鶴花細菌性葉枯病病原菌之引子對 (primer pair)。實驗中使用之引子對是依據葉枯病菌基因組中所選殖出之葉枯病菌專一性核酸探針 (probe) 與16S-23S rDNA基因區間 (spacer region) 之核酸序列而設計。將葉枯病菌 (Xanthomonas campestris pv. dieffenbachiae,簡稱XCD) 之全DNA經內鑑識酵素EcoRI或PstI酵解後,選殖至載體pBluescript SK(-),再配合以XCD及其他與XCD親緣關係相近細菌之全DNA為探針,對各轉形株之重組質體進行差別性雜配反應 (differential hybridization),以篩選對XCD具專一性且適合做為探針之核酸片段,從中篩選出五個對XCD具專一性之轉形株,並進一步評估此等探針之專一性和敏感性,其中以轉形株S10s之嵌入片段 (片段大小為2.3 kb) 所製成之探針具最佳之專一性,遂將該嵌入片段進行核酸解序並依據其序列設計出XCD之專一性PCR引子對S10f-11/S10r-11,其可從XCD DNA模版中,專一地擴增出一大小為705 bp之PCR產物,而不會由其他X. campestris pathovars細菌DNA模版中擴增出任何產物。此外,藉由16S-23S rRNA基因的保守特性,在16S rDNA序列之3''端和23S rDNA序列之5''端設計廣效性引子對Bf1/Br1以增幅出包含有基因區間之585 bp PCR產物並選殖入質體pCRII完成核酸解序,XCD之16S-23S rDNA基因區間核酸序列顯示包含了tRNAAla和tRNAIle的基因序列。利用XCD及其他X. campestris pathovars細菌之16S-23S rDNA基因區間核酸序列比對後依據其序列差異設計出PCR引子對Dif1/Dir1,其可從XCD DNA模版中專一地擴增出一大小為409 bp之PCR產物,而不會由其他測試之X. campestris pathovars細菌、其他種類細菌及火鶴花葉表細菌之DNA模版中擴增出任何產物。PCR引子對S10f-11/S10r-11及Dif1/Dir1的偵測極限為1.5 pg之純化DNA或3-4個XCD細胞。
Two pairs of primers for the polymerase chain reaction-based assay were developed for the detection of the causal agent of anthurium blight, Xanthomonas campestris pv. dieffenbachiae (XCD). One of the primer pairs was synthesized according to the sequence of a cloned DNA probe for XCD, and the other was synthesized according to the sequence of the internal transcribed spacer region between the 16S and 23S rRNA genes of XCD. In order to develop cloned DNA probes for XCD, EcoRI or PstI restriction fragments of genomic DNA of XCD were cloned in plasmid pBluescript SK(-). Five XCD-specific transformants were selected based on the results of differential hybridization using digoxigenin-labeled total DNA of XCD and other closely related pathovars of X. campestris as probes. Cloned DNA inserts from the XCD-specific recombinant plasmid were excised, labeled with digoxigenin, used as probes for dot hybridization and Southern hybridization analyses. The probe from clone S10s (2.3 kb in length) showed the best specificity among the five probes, and the primer pair S10f-11/S10r-11 was thus synthesized according to the nucleotide sequence of the cloned insert. Primer pair S10f-11/S10r-11 could specifically amplify a 705 bp-PCR product using DNA template prepared from XCD but not from other pathovars of X. campestris and other bacterial species. In another approach, universal primers targeting conserved sequences flanking the 3'' end of the 16S and the 5'' end of the 23S rRNA genes were synthesized and used to amplify the 16S-23S rDNA internal transcribed spacer of XCD. A 585 bp-PCR product containing spacer region thus amplified was cloned in pCRII for nucleotide sequence determination. Sequences for tRNAAla and tRNAIle genes were revealed in the 16S-23S rDNA spacer region. Primer pair Dif1/Dir1, synthesized according to the sequence of the spacer region, could effectively amplify a 409 bp DNA fragment from the DNA template prepared from XCD, but not from any tested pathovars of X. campestris, other bacterial species or epiphytic bacteria isolated from anthurium. A minimum of 1.5 pg DNA or 3-4 XCD cells was sufficient to amplify the specific XCD PCR fragments using the primer pairs S10f-11/S10r-11 and Dif1/Dir1.
壹、 前言 1
貳、 前人研究 3
一、 火鶴花細菌性葉枯病病原菌之發現 3
二、 火鶴花細菌性葉枯病病原菌之研究 4
三、 台灣火鶴花細菌性葉枯病及其病原菌之研究 4
四、 火鶴花細菌性葉枯病病原菌之偵測 7
五、 核酸探針之應用 7
六、 聚合酵素連鎖反應在植物病害診斷上之應用 8
七、 16S-23S rDNA spacer region核酸序列在偵測上之應用 10
參、 材料與方法 12
一、 供試菌株來源、培養及保存 12
二、 細菌全DNA之抽取與濃度測定 13
(一) 以phenol/CIAA步驟抽取細菌全DNA 13
(二) 以spin column步驟抽取細菌全DNA 14
(三) DNA濃度及純度測定 15
三、 火鶴花細菌性葉枯病病原菌專一性核酸片段之選殖 15
(一) 葉枯病菌全DNA之選殖 15
(二) 轉形株之篩選與核酸探針之製備 20
(三) 葉枯病菌核酸探針之特性分析 24
(四) 核酸探針嵌入片段之定序及PCR引子對之設計 26
四、 火鶴花細菌性葉枯病病原菌16S-23S rDNA spacer region在偵測上之應用 30
(一) 葉枯病菌16S-23S rDNA spacer region之擴增 30
(二) 聚合酵素連鎖反應產物之選殖及核酸定序 31
(三) 葉枯病菌專一性PCR引子對之設計 34
(四) 葉枯病菌分離株A008專一性PCR引子對之設計 34
五、 PCR引子對專一性和敏感性之測試 35
(一) PCR引子對專一性之測試 35
(二) PCR引子對敏感性之測試 36
肆、 結果 37
一、 火鶴花細菌性葉枯病病原菌專一性核酸片段之選殖 37
(一) 葉枯病菌全DNA之選殖 37
(二)轉形株之篩選與核酸探針之製備 37
(三) DNA探針之特性分析 38
(四) 核酸探針嵌入片段之定序及PCR引子對設計 39
二、 火鶴花細菌性葉枯病病原菌16S-23S rDNA spacer region在偵測上之應用40
(一) 葉枯病菌16S-23S rDNA spacer region之擴增、PCR產物之選殖及核酸定序40
(二) 葉枯病菌專一性PCR引子對之設計 41
三、PCR引子對專一性和敏感性之測試 42
(一)PCR引子對專一性之測試 42
(二)PCR引子對敏感性之測試 43
伍、 討論 45
陸、 中文摘要 53
柒、 英文摘要 55
捌、 參考文獻 57
玖、 圖表 65
拾、附錄 85
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