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研究生:許婷婷
研究生(外文):Hsu,Ting Ting
論文名稱:木瓜畸葉嵌紋病毒台灣品系病害調查、親緣性分析與具感染力轉錄體之構築
論文名稱(外文):A Field Survey, Phylogenic Analysis and Infectious Clone Construction of Papaya leaf-distortion mosaic virus Taiwan Strain
指導教授:江主惠
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:79
中文關鍵詞:木瓜畸葉嵌紋病毒感染力轉錄體多重引子對反轉錄聚合?連鎖反應酵素免疫分析法
外文關鍵詞:Papaya leaf-distortion mosaic virus (PLDMV)infectious transcriptELISAmultiplex RT-PCR
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木瓜輪點病毒(Papaya ringsport virus,PRSV)為主要危害台灣木瓜栽種的病毒,造成嚴重的經濟損失,近年來在田間也發現有木瓜畸葉嵌紋病毒(Papaya leaf distortion mosaic virus,PLDMV)之感染,所以本研究針對 PRSV 及 PLDMV 在台灣的感染情形進行調查。採集來自10個縣市之木瓜樣本,包含來自網室及非網室材料共768個樣本,利用酵素免疫分析法進行病毒偵測,其中有374個樣本(49%)單獨感染 PRSV、27個樣本(4%)單獨感染 PLDMV、134個樣本(17%)為複合感染,剩餘233個樣本(30%)為未感染此兩種病毒之材料。當以酵素免疫分析無法判定其是否感染病毒時,利用多重引子對反轉錄聚合酶連鎖反應方式 (multiplex RT-PCR) 進行偵測,即可依增幅出來的 DNA 片段大小判定其感染病情形。另外,本研究也選殖了15個來自不同地區之 PLDMV 分離株的病毒鞘蛋白基因,利用 NCBI 進行比對,此15個 PLDMV 鞘蛋白選殖株彼此間的核苷酸序列與台灣其他分離株 (TW-DL、TW-TD及TW-WF) 比較有很高的相同度,為94.9%-99%;與日本分離株 (J56P及J179P)有93.7-95.2%相同度,但與瓜類分離株 J199C 相比只有85.1-86.4%。根據 PLDMV 鞘蛋白核苷酸序列進行演化樹分析,則可發現本次所選殖得到的台灣分離株和 TW-TD、TW-KS 及日本之 PLDMV 歸屬於不同群組。另外,為了測試台灣 PLDMV 木瓜分離株是否也能感染瓜類,分別將來自農試所試驗田及大里之 PLDMV 接種於胡瓜及刺角瓜中,並以酵素免疫分析法及西方墨點法偵測病毒感染,結果發現 PLDMV 均無法感染胡瓜,感染刺角瓜的比例只有8.8%且無明顯病徵。之前本實驗室已將大部分 PLDMV 大里分離株 (TW-DL)的基因體解序完成,但最5'端之序列仍然未知,因此本研究再利用5'RACE 來獲得 PLDMV-DL 5'端序列,並將其全長度及5'UTR 之核苷酸序列與網路 PLDMV TW-KS 及 J56P 進行比對,結果全長度基因體之相同度約為95%,而5'UTR 序列為84%-87%。在 PLDMV 具感染力轉錄體的構築方面,選殖七個互相重疊可以涵蓋整個 PLDMV 基因體的質體,分別為 p35S-5PL426、p5PL-2764、pPL2680NarI-4361 、pPL4247-6604、pPL5761-8298、pPL8150-9810及 pPL9528-PolyA-NotI。目前已完成 p35SPL2711-9779NotI 之構築,此質體中的35S 啟動子後面接有 PLDMV 第1到第2711個核苷酸與第9779至3'端 poly A 的 DNA 片段,只需再將第2680-9810的片段剪切黏合,即可完成 PLDMV 全長度之構築。
Papaya ringspot virus (PRSV) causes the most economy loss in papaya production of Taiwan. Recently, the Papaya leaf-distortion mosaic virus (PLDMV) was also found. The infection rate of both viruses on papaya are uncertain. In this study, we investigated the PRSV and PLDMV distribution in Taiwan. Total 768 samples of papaya were collected from net-houses and field in papaya growth area, including MiaoLi, Changhua, Yunlin, Nantou, Chia-yi, Tainan, Kaohsiung, Pingtung, Taitung, and Hualien. According to Enzyme-Linked Immunosorbent Assay (ELISA), 53% of samples were only infected with PRSV, 4% were only infected with PLDMV, 17% were co-infected with PRSV and PLDMV, and 30% were undetectable. Multiplex RT-PCR for the specific detection of PRSV and PLDMV was used to identify the ELISA uncertain samples. The amplified DNA fragments of 378 bp and 564 bp indicated the infection of PRSV and PLDMV, respectively. The coat protein genes of 15 PLDMV samples that amplified by RT-PCR were further cloned. Sequence analysis of these CP genes showed 97.7 to 99.7% of nucleotide identities with each other, and 94.9-99% identity with other Taiwan isolates(TW-KS, TW-DL, and TW-TD. Moreover, these CP genes showed 93.7-95.2% identity with Japan strain(J56P and J179P)and much lower sequence identity(85.1-86.4%)with J199C strain which only infected cucurbit specise. Phylogenetic tree of 15 PLDMV CP gene is shown in differen clade of TW-TD、TW-KS and Japan isolates. To determine if the PLDMV-P Taiwan isolates could infect cucumber plants, two PLDMV isolates were inoculated into Cucumis metuliferus Acc.2459 and Cucumis sativus and the infectivity of the virus was detected by ELISA and western blotting analysis. Only 8.8% of symptomless C. metuliferus showed PLDMV infection. In previous study, the full-length PLDMV DL sequence was complete except for the nucleotides near the 5' end. 5'RACE was performed to obtain the 5'end sequence. Comparison of the full-length sequence of DL isolate with PLDMV TW-KS and J56P showed 94.5% and 94.6% of nucleotide identity, respectively. When comparing with the 5'UTR region, the identity to PLDMV TW-KS and J56P was 87.4% and 84.4%, respectively. With the PLDMV-DL complete sequence, an infectious transcript can be constructed. Seven PLDMV clones, including p35S-5'426, pPL5'-2764, pPL2680NarI-4361, pPL4247-6604, pPL5761-8298, pPL8150-9810, and pPL9528-3'NotI, with overlapping DNA fragments to each other were obtained by RT-PCR. These PLDMV DNA fragments were used to replace the sequence in 35S-PRSV infectious clone. A plasmid p35SPL2711-HA-9779NotI was obtained that contained the 35S promoter sequence followed by the 5'end of PLDMV nt 1 to 2711 and the 3'end of nt 9779 to the poly(A) tail. Finally, by introducing the PLDMV DNA fragment of nt 2680 to 9810 into p35SPL2711-HA-9779NotI, the full-length genomic sequence of PLDMV will be complete.
封面內頁
簽名頁
授權書 iii
中文摘要 iv
英文摘要 vi
誌謝 viii
目錄 ix
圖目錄 xii
表目錄 xiii

1. 前言 1
1.1 木瓜之特性 1
1.2 木瓜之病毒病害 1
1.3 馬鈴薯Y群病毒之特性 2
1.4 木瓜輪點病毒(PRSV)在台灣之發生與防治策略 3
1.5 木瓜畸葉嵌紋病毒(PLDMV)之發現及其特性 5
1.6 病毒具感染力轉錄體之構築 7
1.7 實驗目的 10
2. 材料與方法 12
2.1 台灣木瓜輪點病毒及木瓜畸葉嵌紋病毒病害調查 12
2.1.1 疑似染病木瓜材料之田間採集 12
2.1.2 罹病木瓜材料之保存 12
2.1.3 酵素連結免疫分析(Enzyme-Linked Immunosorbent Assay, ELISA)偵測木瓜病毒病害 13
2.2 利用多重引子對以反轉錄聚合酶連鎖反應(multiplex RT-PCR)進行病毒檢測 14
2.2.1 核醣核酸萃取 15
2.2.2 Multiplex RT-PCR 步驟 16
2.3 繪製田間病害調查採集之木瓜衛星定位 17
2.4 PLDMV 宿主範圍測試及西方墨點法 17
2.4.1 西方墨點法(Western blotting)偵測植物中之病毒 17
2.4.1.1 植物蛋白質之粗萃 (Protein extraction) 17
2.4.1.2 SDS-PAGE 之配置 18
2.4.1.3 蛋白質電泳之進行及電轉印 (Electrophoresis and electroblotting) 19
2.4.1.4 血清雜合與呈色(Serum hybridization and color       developement) 20
2.5 PLDMV 鞘蛋白基因解序與演化分析 21
2.5.1 PLDMV 鞘蛋白序列之選殖 21
2.5.2 病毒鞘蛋白 DNA 送至 TA 載體 21
2.5.3 製備勝任細胞以及質體的轉型作用 22
2.5.4 質體 DNA 之快速篩選(Quick screening) 23
2.5.5 質體 DNA 之純化 23
2.5.6 核苷酸序列分析 24
2.5.7 鞘蛋白序列之比對與演化關係分析 24
2.6 PLDMV 5'端序列之選殖 25
2.6.1 第一股 cDNA 的合成 25
2.6.2 以 S.N.A.P. Column 純化第一股 cDNA 26
2.6.3 將 dCTP 加至第一股 cDNA 的3'端 (TdT Tailing of cDNA) 26
2.6.4 針對 dC-tailed cDNA 進行 PCR 26
2.7 PLDMV 全長度 cDNA 之構築 27
2.7.1 含35S 啟動子與 PLDMV 5'端質體之構築 27
2.7.2 涵蓋 PLDMV 其它區域之 DNA 選殖 29
2.7.3 PLDMV 2680-9810序列之構築 30
2.7.4 PLDMV 全長度 DNA 片段之連接 31
3. 結果 33
3.1 台灣木瓜病毒病害調查 33
3.2 台灣木瓜畸葉嵌紋病毒鞘蛋白基因之相同度與演化分析 36
3.3 PLDMV 大里 isolate 之5'端核苷酸之選殖與解序 37
3.4 瓜類寄主感染病毒之測定 37
3.5 PLDMV 全長度 cDNA 之構築 38
4. 結論 40
參考文獻 66
附錄 74









圖目錄

圖1、以 PCR 方式黏合35S 啟動子與 PLDMV 5'端序列。 46
圖2、涵蓋 PLDMV 基因體之大片段 DNA 構築 47
圖3、PLDMV 全長度基因體之構築 48
圖4、田間網室栽培之木瓜,疑似受病毒感染之病徵 50
圖5、酵素免疫分析法 (ELISA)及多重引子對反轉錄聚合酶連 鎖反應 (multiplex RT-PCR)偵測 PRSV 及 PLDMV 51
圖6、田間網室及非網室種植之木瓜植株感染 PRSV 及 PLDMV 之比例圖 52
圖7、台灣各地區採集共768個木瓜樣本,依其染病情形所繪得之衛星分布圖 53
圖8、23個不同 PLDMV isolate 鞘蛋白核苷酸序列之演化關係分析 54
圖9、以西方墨點法偵測接種在瓜類上的 PLDMV 病毒 55
圖10、含 PLDMV DNA 序列的質體,以 HindIII 及 EcoRI 剪 切後之電泳圖,以確認其選殖之正確性 56








表目錄

表1、構築含35S 啟動子及涵蓋全長度 PLDMV 基因體時所使 用的引子對 57
表2、台灣田間網室之木瓜 PRSV 及PLDMV 病毒病害調查統 計 58
表3、台灣各鄉鎮之田間非網室木瓜病害調查統計 59
表4、本研究選殖之15個 PLDMV 分離株之鞘蛋白與日本分離 株及其他台灣分離株之核苷酸序列比對 63
表5、台灣 isolate PLDMV-DL 與其他來源的 PLDMV 核苷酸 序列相同度之比對 64
表6、大里 isolate (TW-DL)及農試所試驗田分離之 PLDMV 機 械接種 65
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