劉瑞芬臺灣大學：植物病理與微生物學研究所黎忠祥Lai, Chong-CheongChong-CheongLai2007-11-272018-06-292007-11-272018-06-292006http://ntur.lib.ntu.edu.tw//handle/246246/58017Phytophthora parasitica是重要植物病原菌，具有廣泛的寄主範圍且能感染超過72屬的植物。P. parasitica為異宗交配的疫病菌，其A1與A2配對型的菌株在台灣皆可被找到。疫病菌的有性生殖不僅在遺傳物質的交換扮演重要的角色，同時也能產生耐受惡劣環境的存活構造。長期以來學者們對疫病菌配對型的決定甚感興趣，然而在分子層次的了解並不多。 98130A1與98130A2這兩株疫病菌進行反轉錄跳躍子去氧核糖指紋分析發現兩者的雜合圖譜幾乎一致而只有少數的差異，顯示兩者的遺傳背景非常近似。以32組引子對組合進行增幅性片段長度多型性分析(AFLP)，發現大部分引子對所增幅出來的增幅性片段長度多型性圖譜非常的類似，然而還是可以得到8個僅出現在單一配對型之差異性條帶。序列分析發現3個差異性片段具有Rumex acetosa Y 染色體專一性重覆性序列之同源性片段，然而仍需要進一步的研究來確認其是否與配對型有關。穩定環境下培養3天的98130A1與98130A2疫病菌，進行互補核酸增幅性片段長度多型性(cDNA-AFLP)分析，利用40組引子對兩者進行分析發現所增幅出來的條带形式非常相似，但是仍可以得到44個具差異性表現衍生片段。cDNA-AFLP的分析結果發現，A15T13-2 UTDF在98130A2菌株有較高的表現，進一步利用北方雜合分析與即時反轉錄聚合酶連鎖反應(real-time RT-PCR)，確認了cDNA-AFLP結果的正確性。增加A1與A2菌株的數量進行另一次北方雜合分析，A2菌株表現量也高於A1菌株，這說明A15T13-2 UTDF在P. parasitica A2菌株有較高表現量的現象，可能具有共通性。A15T13-2 UTDF經由序列比對發現其與Danio rerio的aquaporin 3具有很高的相似度，然而這個基因與配對型的決定是否具有相關性，仍需要進一步的實驗證實。Phytophthora parasitica is an important plant pathogen with a broad host range capable of infecting more than 72 plant genera. P. parasitica is heterothallic with A1 and A2 mating types have been isolated in Taiwan. The sexual reproduction of Phytophthora plays an important role not only in genetics exchange, but also as a survival structure. In spite of numerous efforts made to determine the mating type of Phytophthora not much is known at the molecular level. Retroposon-base DNA fingerprint of 98130/A1 and 98130/A2 isolates of P. parasitica were analyzed.The hybridization pattern of 98130/A1 was almost the same as 98130/A2 with the exception of few bands, indicating that these two strain were isogenic. In AFLP experiment, a total 32 AFLP primer sets were analyzed for their ability to distinguish isolates of opposite mating type. Although the AFLP patterns were similar in most cases, 8 bands were found only in one mating type but not the other. Sequence analysis showed three of to containe homologous sequences of Rumex acetosa Y chromosome specific tandem repeat however, further study is needed to confirm its genetic link to mating type. In cDNA-AFLP experiment, a total of 40 combination primer sets were analyzed for their ability to distinguish between 98130/A1 and 98130/A2 in stable environmental conditions. Although the cDNA-AFLP patterns were similar in most cases but there did exit 44 differential transcription derivate fragments. The transcription derivate fragment A15T13-2 revealed by cDNA-AFLP expressed higher in 98130/A2 than 98130/A1. The validity of this result was comfirmed by northern hybridization and real-time PCR. The performance of northern hybridization with increased number of A1 and A2 isolates showed similar result, indicating that the phenomenon of higher expression of A15T13-2 in A2 isolates of P. parasitica is universal. A15T13-2 showed high similarity with Danio rerio aquaporin3 by sequence analysis. These differential transcription derivate fragments need further study to confirm its linkage with either mating type determination or mating process.中文摘要………………………………………………………………………….......I Abstract………………………………………………………………………………III 壹、 前人研究...............................................................................................................1 一、 疫病菌分類地位.................................................................................1 二、 疫病菌的基本型態與特徵…………………………………………….1 三、 發病生態...................................................................................................2 四、 疫病菌有性世代的相關研究………………………………………….3 五、 Phytophthora parasitica的研究………………………………………6 六、 台灣發生現況與危害…………………………………………….......10 貳、材料與方法…………………………………………………………………...11 一、 菌株來源……………………………………………………………...11 二、 菌株配對型確認……………………………………………………...11 三、 DNA之製備…………………………………………………………..12 四、 G2ty-1 DNA指紋分析......................................................................13 1. Genomic DNA酵解以及瓊脂電泳分析…………………………..13 2. DNA毛細作用轉漬法……………………………………………..13 3. G2ty-1探針與質體小量製備……………………………………...14 4. DIG (digoxigenin)標的G2Ty-1探針……………………………...14 5. G2Ty-1之核酸定序與序列分析…………………………………..15 6. 雜合前置反應 (Prehybridization)………………………………...16 7. 雜合反應 (Hybridization)…………………………………………16 五、 Amplify Fragment Length Polymorphism (AFLP)…………………..17 1. Genomic DNA酵解與接合………………………………………..17 2. 前增幅反應 (Pre-selective amplification)………………………...17 3. 選擇性增幅反應 (Selective amplification)…………………….....18 4. 定序電泳膠的配製與電泳分析.....................................................18 5. 硝酸銀染色 (silver stain)……………………………………….....19 6. 差異性DNA片段的回收與增幅………………………………….19 7. 差異性DNA片段擴增產物選殖………………………………….20 8. 選殖株篩選 colony PCR…………………………………………..21 9. 質體製備……………………………………………………………21 10. 質體小量製備………………………………………………………22 11. 定序反應……………………………………………………………23 12. 酒精沈澱……………………………………………………………23 13. 定序與序列分析……………………………………………………23 六、 cDNA-AFLP………………………………………………………....24 1. 利用Trizol抽取P. parasitica total RNA………………………..24 2. 純化Poly (A)+ RNA………………………………………………24 3. First strand cDNA合成…………………………………………...25 4. Second strand cDNA合成………………………………………..25 5. Double strand cDNA酵解與接合................................................26 6. 前增幅反應 (Pre-selective amplification)……………………....26 7. 選擇性增幅反應 (Selective amplification)……………………..27 8. 定序電泳膠的配製與電泳分析………………………………….27 9. 硝酸銀染色 (silver stain)………………………………………..27 10. 差異性cDNA片段的回收與增幅..............................................27 11. 差異性cDNA片段的選殖與定序...............................................27 七、 北方雜合分析 (Northern hybridization)……………………………..28 1. Total RNA甲醛變性瓊脂電泳....................................................28 2. RNA毛細作用轉漬法.................................................................28 3. DIG標的cDNA-AFLP差異性片段……………………………..29 4. 雜合前置反應 (Prehybridization)……………………………….29 5. 雜合反應 (Hybridization)………………………………………..29 八、 Real-time reverse transcription polymerase chain reactions…….….31 1. DNase I處理與cDNA之合成……………………………………..31 2. Real-time PCR之相對定量………………………………………...32 3. 定量結果之分析...............................................................................32 參、結果……………………………………………………………………………33 1. Retroposon-based DNA fingerprint analysis………………........33 2. AFLP……..…………………………………............................33 3. cDNA-AFLP…………..…………………………….............36 肆、討論...............................................................................................................44 1. AFLP………………………………………………………………44 2. cDNA-AFLP………..………..…………………………………...46 伍、結論……………………………………………………………………………52 陸、圖表................................................................................................................54 柒、參考文獻.......................................................................................................72 捌、附錄…………………………………………………………………………….912857537 bytesapplication/pdfen-US疫病菌增幅性片段長度多型性Phytophthora parasiticaamplified fragment length polymorphismotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58017/1/ntu-95-R92633006-1.pdf