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研究生:柯曉芝
研究生(外文):Ko,Hsiao-Chih
論文名稱:甘薯蔟葉病病原似菌質體核酸探針之製備與應用
論文名稱(外文):Development and application of DNA probe for the mycoplasmalike organism(MLO) associated with sweetpotato witches' broom
指導教授:林長平林長平引用關係
指導教授(外文):Lin,Chan-Pin
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病蟲害學系
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:1993
畢業學年度:81
語文別:中文
論文頁數:83
中文關鍵詞:甘薯簇葉病似菌質體核酸探針
外文關鍵詞:sweetpotato witches' broommycoplasmalike organismDNA probe
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本研究乃針對甘薯簇葉病(SPWB)之病原似菌質體(MLO)以基因重組技術進
行其核酸探針之研製及利用。將純化之 MLO DNA 並與載體黏合後進行轉
形反應,獲得具有嵌入 DNA 重組質體之轉形菌株,藉由點漬染反應篩選
對 SPWB 罹病植物 DNA 呈正反應的轉形菌株, 由其中分離出 MLO DNA
片段製成探針。在專一性測試中,針對不同病害之 MLO DNA 分別利用點
雜配法進行分析,在這十四個探針中有二個探針 SPWB16 (5.8kb) 及
SPWB27 (3.5kb) 只對由 SPWB-MLO 罹病甘薯及日日春抽取之全 DNA 具有
專一性反應,而對嫁接至日日春之花生簇葉病,絲瓜簇葉病, 細花矮牽
牛簇葉病,泡桐簇葉病,榆樹黃萎病,翠菊黃萎病, 或由田間採集之水
稻黃萎病,竹子小葉病等罹病植物全 DNA 則無雜配反應訊號。另外十二
個探針(SPWB5, 2.0kb;SPWB8,2.5kb;SPWB12,4.3kb;SPWB15,1.6kb
;SPWB25,1.1kb; SPWB28,1.1kb; SPWB36,5.3kb; SPWB54,1.2kb
; SPWB59,3.0kb; SPWB62,5.2kb; SPWB73 ,3.0kb;SPWB99 ,2.3
kb) 則除了甘薯簇葉病外,亦與花生簇葉病有雜配反應。由上述選出之探
針進行南方氏轉漬及雜配,則可對這二種 MLO 進行區別。利用組織轉印
法,可以觀察在病株中 MLO 存在位置。利用專一性探針進行敏感性測試
,當 SPWB-MLO 罹病日日春之全 DNA 稀釋至 0.05ng 時,仍可被有效被
偵測到,而 SPWB-MLO 罹病甘薯之全 DNA 稀釋至 0.3ng 時,亦可被偵測
。將探針 SPWB54 之 DNA 片段經核酸定序分析,挑選部分序列合成引子
對,針對上述 SPWB- MLO 罹病植物之全 DNA 進行該 DNA 片段增殖 ,其
他似菌質體罹病植物及健康植物 , 皆無該 DNA 之增殖於 35 個循環反應
下,對 SPWB-MLO 罹病之日日春或甘薯全 DNA,則 DNA 稀釋至 10pg 時
,仍能有效增殖該片段 DNA 。由以上結果顯示,本研究中所研製成功之
核酸探針,於病原偵測,病害診斷,病原鑑定等相關研究上,提供了極為
有用之研究工具。

Fourteen probes were selected and used in dot-hybridizations
and Southern hybridization analyses for the differentiation of
SPWB-MLO from various MLOs.Two probes SPWB16,SPWB27 hybridized
with DNAs from SPWB-MLO affected plants but not with DNAs from
healthy plants and plants infected with other MLOs. Twelve
other probes (SPWB5;SPWB8;SPWB12;SPWB15;SPWB25;SPWB28;SPWB36;
SPWB54; SPWB59;SPWB62;SPWB73;SPWB99) hybridized with DNA from
plants affected with SPWB, PNWB but not with DNA from healthy
plant and other MLOs. The specific probes detected the DNA from
periwinkle infected with SPWB-MLO to 0.05-0.1ng and the DNA
from sweetpotato infected with SPWB-MLO to 0.3-0.7ng
effectively. In the Southern hybridization analyses,nine of the
probes though crossreacted to PNWB-MLO in dot-hybridization can
differentiate the SPWB-MLO from PNWB-MLO. A polymerase chain
reaction(PCR)method has been applied in the detection of SPWB-
MLO. Primers were selected from SPWB54. Amplification of SPWB-
MLO DNA with 1.1 kb in length had been achieved with DNA from
SPWB,PWB-MLO affected plants but not with DNA from healthy
plants and plants infected with other MLOs after 25 cycles of
reactions. To evaluate the minimal amount of DNA required for
the amplification of MLO-specific DNA ,an MLO-DNA fragment was
obtained after amplification of 10pg of total DNA from diseased
plants after 35 cycles of reactions, respectively.

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