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研究生:卜樂妙
論文名稱:腐霉菌粒線體核酸探針的選殖與應用
論文名稱(外文):Selection and Application of Species-specific DNA Probes for Plant Pathogenic Pythium
指導教授:趙維良趙維良引用關係
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1994
畢業學年度:82
語文別:中文
論文頁數:110
中文關鍵詞:腐霉菌
外文關鍵詞:Pythium
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腐霉菌 (Pythium) 是國內水耕及土壤栽培作物的重要病原菌,其鑑定向以形態觀察為主,有的菌種間因形態相近而易導致鑑定錯誤,連帶影響病害診斷與防治。本研究係利用 RFLP技術建立實用的鑑定方法,以輔助傳統的方法;此外由粒線體核酸片段選殖專一性核酸探針,以供偵測病原菌之用。研究結果顯示,測試的二十個腐霉菌種,其種間粒線體核酸限制酵素切割片段多型性 (RFLPs) 的相似度大多低於 30%。由粒線體核酸選殖菌種間同源性高的核酸片段作為探針,以比較菌株之間的種系相關性 (phylogenetic relationships),其中探針 pJM73 可使測試的十九株 P. aphanidermatum 菌株呈現兩種核酸雜合圖譜,顯示其粒線體核酸有種內變異情形存在;核酸探針 pJMM19-3、pJM19-18、pJM19-29 及 ML-Ⅱ 與十九個菌株的全量核酸限制酵素切割片段進行雜合反應,所呈現的核酸雜合圖譜進行層系分群分析並作出樹狀圖,結果顯示十九個腐霉菌種可區分為三群,群與群之間的相似係數百分比最高不超過 30%。此外pJM19-29 及ML-Ⅱ 配合限制酵素 MspI 及 HindⅢ,所呈現的核酸雜合圖譜可將測試的十九個菌種做區分。專一性核酸探針的選殖方面,pJM73-2 帶有 P. aphanidermatum 粒線體核酸經HindⅢ-MspⅠ切出的 1.55kb 核酸片段;pJD4 及 pJD34 帶有 P.deliense 粒線體核酸經 PstⅠ-XbaⅠ切出的核酸片段,前者為 0.45 kb而後者為 1.2kb。將這三個核酸探針與二十個腐霉菌種的全量核酸,於68℃進行點漬雜合測試,結果顯示 pJM73-2 與 P.aphanidermatum 核酸產生強烈的雜合反應,與 P. deliense 核酸有微弱的反應,與其他測試的腐霉菌種不產生雜合作用;而pJD4 及 pJD34 則僅與 P. deliense 核酸產生明顯的雜合訊號。此外三個核酸探針與五株 Phytophthora、八株 Fusariumoxysporum 等植物病原真菌的核酸,以及水白菜、空心菜、大胡瓜、小黃瓜、洋香瓜、莢碗豆等供試作物的核酸,亦不產生交互反應。經點漬法測試,專一性核酸探針的靈敏度約為20-50ng 的全核酸量。於實驗室將 P. aphanidermatum 接種到植株,pJM73-2 可由 200ng 的病株全量核酸中偵測病原菌的存在。


The genus Pythium is one of the most important plant pathogenic fungi. Traditionally, species identification in Pythium is based upon morphological features including charactertistics of asexual reproduction and sexual reproduction. However, many species of Pythium possess similar morphological features and some even failed to produce reproductive structures, hence resulting in the difficulty in their identification. In this study, molecular technology was used to facilitate the identification and detection of different Pythium species. Mitochondrial DNAs of 15 different Pythium species were isolated, digested with restriction enzyme (HindⅢ) and electrophoresed. The DNA fragment banding patterns of these species showed distinct variation. Furthermore, total DNAs of 20 Pythium species were also extracted and digested with restriction enzymes including HindⅢ, MspⅠ, HaeⅢ, and PstⅠ. The resulted DNA fragments were then electrophoresed and transfered to nitrocellulose membrane. The membranes were then hybridized with non-radiactive-labeled probe prepared from mtDNA of P. torulosum. The similarity of hybridization patterns were calculated based on Jaccard coefficient and the results indicated that the similarity coefficients among species were generally lower than 30%. To compare phylogenetic relationships among Pythium species, common DNA fragments from Pythium were isolated and served as probes. Probes pJM19-3, pJM19-18, pJM19-29, and ML-Ⅱ were cloned from P. ultimum mtDNA fragments and hybridized with the total DNAs of 19 Pythium species. The results showed high homology among these species. When these probes were used to hybridize with the restriction fragments of total DNAs of 19 Pythium species by HindⅢ, MspⅠ, and RsaⅠ, the resulting patterns were further processed by hierarchical cluster analysis to construct a phylogenetic dendrogram. It showed that 19 Pythium species tested could be divided into three distinct groups, and the similarity coefficients among these groups were less than 30%. However, when total DNAs were digested with MspⅠ or MspⅠ-HindⅢ and hybridized with probes pJM19-29 and ML-Ⅱ, distinct hybridization patterns were observed. Furthermore, for quick detection and identification of pathogenic Pythium, specific DNA probes had been selected. A 1.55kb fragment (pJM73-2) was obtained from P. aphanidermatum mtDNA, and a 0.45kb clone (pJD4) and a 1.2kb clone (pJD34) were obtained from P. deliense mtDNA. Probe pJM73-2 could strongly hybridized with P. aphanidermatum total DNA but slight cross reaction with P. deliense total DNA was also detected. As for probes pJTD4 and pJD34, they only hybridized with P. deliense total DNA. When the specificity of these probes was examined, total DNAs from 5 Phytophthora isolates, 8 Fusarium isolates, and 6 plant species were used. The result showed no hybridization signals could be detected between these probes and DNA samples. The amount of test sample DNAs required for positive hybridization for these species-specific probes were about 20-50 ng of total DNA. In the laboratory infection, probe pJM73-2 could detect P. aphanidermatum in 200 ng total DNA of infected plants.

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