Application of Monoclonal Antibodies and Cloned DNA Probes for the Detection and Differentiation of Phytopathogenic Mycoplasmalike Organisms

單元抗體及DNA探針在植物病原似菌質體之鑑定及偵測上之應用

林長平(Chan-Pin Lin) ； 沈偉强(Wei-Chiang Shen) ； 柯曉芝(Hsiao-Chih Ko) ； 張富林(Fu-Lin Chang) ； 游燕伶(Yen-Ling Yu) ； 陳妙帆(Miaw-Fan Chen) ； 吳鳳儀(Fen-Yi Wu)

《植物病理學會刊》 2卷3期 (1993/09) Pp. 161-168

https://doi.org/10.6649/PPB.199309_2(3).0007

摘要

針對甘藷簇葉病、花生簇葉病與水稻黃萎病利用融合瘤技術及DNA選殖技術，成功地研製出對抗甘藷簇葉病與水稻黃萎病病原似菌質體之單元抗體，以及對抗甘藷簇葉病、花生簇葉病與水稻黃萎病病原似菌質體之DNA探針。對抗甘藷簇葉病病原似菌質體之單元體顯示了甘藷簇葉病似菌質體、花生簇葉病病原似菌質體及在澎湖島上發現的三種雜草簇葉病之病原似菌質體間具有血清相關性。而對抗水稻黃萎病病原似菌質體之單元抗體則只專一地對水稻黃萎病病原似菌質體具血清反應，此單元抗體可在西方漬染反應中認識二條大小為16 kD及41kD屬於水稻黃萎病病原似菌質體之多胜呔。在DNA探針之研製上，利用對抗甘藷簇葉病病原似菌質體之DNA探針在南方漬染反應中可依其雜配反應圖譜而明確地鑑別出上述具血清相關性之似菌質體。此些甘藷簇葉病病原似菌質體之DNA探針，其大小介於0.5-7kb，其對病甘藷之全DNA的偵測能力為0.3-0.7ng，而對日日春病株全DNA則可偵測至0.05-0.1ng。對抗花生簇葉病病原似菌體之DNA探針其大小介於0.5-4kb，亦能明確地鑑別不同病原似菌質體。而在對抗水稻黃萎病病原似菌質體之DNA探針之研製上，共獲得十二株特異之探針，其大小介於1.0-5.5kb，其中並包括依大小為4.3kb的質體探針，此些探針對病水稻株之全DNA偵測能力為3-13ng。

關鍵字

DNA探針 ； 單元抗體 ； 似菌質體

並列摘要

Toward mycoplasmalike organisms (MLOs) associated with sweet potato witches' broom (SPWB), peanut witches' broom (PWB), and rice yellow dwarf (RYD), hybridoma techniques and genetic cloning techniques have been applied in the production of pathogen-specific monoclonal antibodies (MAs) and cloned DNA probes. In the generation of specific MAs and DNA probes for the disease detection and pathogen differentiation, specific monoclonal antibodies for SPWB-MLO, and RYD-MLO along with cloned DNA probes for SPWB-MLO, PWB-MLO, and RYD-MLO had been successfully developed. Monoclonal antibodies against SPWB-MLO revealed the serological relatednesses among SPWB-MLO, PWB-MLO, and MLOs associated with three different witches' broomed plants found on the Penghu Islands. MAs for RYD-MLO can specifically reacted with RYD-MLO only. The antibody recognized two polypeptides, 16 kD and 41 kD. in Western blotting. Further investigations were carried out by employing cloned DNA probes for the differentiation of the serologically related MLOs associated with SPWB, PWB and witches' broom found in various weeds. When cloned DNA probes for SPWB-MLO were applied in the Southern hybridization, distinct hybridization patterns were obtained though they did cross hybridize to some serologically related MLOs in dot-hybridization. For SPWB-MLO specific DNA probes, the sizes for cloned DNA fragments ranged from 0.5 to 7 kb, and the minimal amounts of DNA detectable in dot-hybridization tests were around 0.3 to 0.7 ng toward total DNA from diseased sweet potato and 0.05 to 0.1 ng toward total DNA from SPWB-MLO affected periwinkles. For PWB-MLO specific DNA probes, the sizes of cloned DNA fragments ranged front 0.5 to 4.0 kb. Sonic of these PWB-MLO probe did cross react with serologically related SPWB-MLO. For RYD-MLO specific DNA probes, 12 specific probes including one plasmid-probe were obtained and reacted specifically only with RYD-MLO. The sizes of cloned DNA fragments ranged from 1.0 to 5.5 kb. The minimal detectable amounts of DNA from RYD-affected rice were around 3-13 ng when RYD-MLO DNA probes were applied in dot-hybridization.

並列關鍵字

DNA probe ； monoclonal antibody ； mycoplasmalike organism