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頁籤選單縮合
題 名 | Botrytis Cinerea及B. Elliptica偵測用分子標記之建立=Establishment of Molecular Markers for Detection and Diagnosis of Botrytis Cinerea and B. Elliptica |
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作 者 | 陳隆鐘; 陳天枝; 陳惠玲; 葉瑩; | 書刊名 | 植物病理學會刊 |
卷 期 | 7:4 1998.12[民87.12] |
頁 次 | 頁177-188 |
分類號 | 433.4 |
關鍵詞 | 灰黴病菌; 粒線體核酸探針; 聚合; 連鎖反應; Botrytis cinerea; B. elliptica; DNA probes; Polymerase chain reaction analysis; |
語 文 | 中文(Chinese) |
中文摘要 | 將Botrytis cinerea菌株WFFr103與B. elliptica菌株EPL803之粒腺體核酸經限 制酵素EcoRI處理後,分別與載體pBluescript SK+接合,再轉型至E coli DH5α菌株,各 構築一基因庫,並自轉殖株回收之mtDNA片段製備成的核酸探針與其餘菌株之全DNA進行點 雜配反應,測試結果以核酸探針pBE83-8(0.9kb)可偵測至只含10 mg之灰黴病菌全量核 酸之高敏感度,核酸探針pBE83-4B(1.2kb)與所有供試B. elliptica菌株之核酸同源性最高 ,皆有反應出現。另以隨機增幅核酸多型性分析,篩選出引子OPB-17,其增幅灰黴病菌DNA 時,皆有專一性300 bp、600 bp及800 bp左右之條帶產生,並進一步將此些片段進行轉殖 及製備成探針pBCE30、pBCE60及pBCE80。以南方氏核酸雜合反應分析,其與Botrytis cinerea和B. elliptica菌株皆有反應產生,並分別進行核酸序列解序設計引子對PB30-1F/ PB30-1F/PB60-1R和PBCE80-1F/PB80-1R,其中引子對PB80-1F/PB80-1R能專一性自B. cinerea 增幅出890 bp的條帶,和從B. elliptica增幅出381 bp的條帶,但對Sclerotium rolfsii 與Mucor sp.則無任何產物產生。 |
英文摘要 | Botrytis cinerea is a widespread pathogen producing infections on more than 200 bosts. Hosts attack can occur before harvesting or later during transport or storage. And B. elliptica causal agent of leaf blight, inflicts serious losses cut flowers of lily in Taiwan. However, B. cinerea and B. elliptica have become one of the most important threats to the production, import and export of cut flowers and some important crops. However, detection of the fungus at the latent stage befor packing for transitting is diffcult because time consuming and tedious by classical methods. In this study we describe the development of DNA probe and polymerase chain reaction analysis to detection of B. cinerea and B. elliptica. A genomic library of mitochondrial DNA, digested with EcoRI restriction enzyme from isolates of WFFr103、EPL803 of B. cinerea and B. elliptica was constructed in the vector of pBluescript (SK+), and transformed into E. coli DH5 α. Randomly selected recombinant clones designated as pBE83-8 (0.9 kb)、pBE83-4B(1.2 kb)、pBE83-7(1.1 kb)、pBC26-9(1.2 kb)、pBC29-9(0.7 kb) and pBC29-2(1.2 kb) were nonradioactive labelled with NEBlot-Phototope . The sensitivity of DNA probe Be 83-8(0.9 kb) is highest up to 10 �瓊gg among all DNA probes from dot-blot hybridization analyses with total DNA of B. cinerea and B. elliptica. But the DNA probe of Be 83-4B(1.2 kb) hybridized with all isolates of B. cinerea and B. elliptica. According to RAPD band patterens analyses with primer OPB-17 to ampify DNA of B. cinerea and B.elliptica have the 300 bp、600 bp and 800 bp fragments in amplified DNA after gel electrophoresis. These 300 bp 、600 bp and 800 bp fragments transformant clones were prepared for DNA probes, as pBCE30、pBCE60 and pBCE80. And the high specificity of DNA probes of pBCE30 、pbCE60 and pBCE80 were proved with Southern hybridzation with all isolates of B. cinerea and B. elliptica. For sets of primers of PB30-1R/PB30-1F、PB60-1R/ PB60-1F and PB80-1F/PB80-1R were developed from the nucleotide sequences analysing of insert DNAs in pBCE30、pBCE60 and pBCE80, respectively, useing total DNAs of B. cinerea、B. elliptica and another check fungus isolates as templates in PCR assay. The PB80-1F/PB80-1R primers set specifically amplified 890 bp DNA fragments from DNA of B. cinerea, and amplified 381 bp DNA fragments from DNA of B. elliptica but not DNA from any another check isolates as Sclerotium rolfsii and Mucor sp. Using of this highly specific and reliable detection could prove valuable for regulatory, epidemiological and ecological studies. |
本系統中英文摘要資訊取自各篇刊載內容。