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研究生:王映方
研究生(外文):Yin-Fan Wang
論文名稱:以人工重組感染性選殖株探討木瓜輪點病毒基因變異對致病性之影響
論文名稱(外文):The study of genomic variation of Papaya ring spot virus (PRSV) affecting its pathogenicity with artificially recombinant infectious clones
指導教授:洪挺軒
口試委員:沈湯龍陳穎練
口試日期:2015-06-12
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理與微生物學研究所
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:2015
畢業學年度:103
語文別:中文
論文頁數:74
中文關鍵詞:木瓜輪點病毒轉基因感染性選殖株P1基因
外文關鍵詞:Papaya ring spot virustransgenic papayainfectious cloneP1 gene
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木瓜為台灣重要果樹,感染木瓜之病害中以木瓜輪點病毒(Papaya ring spot virus, PRSV)引起的木瓜輪點病(Papaya ring spot)最具破壞性。此病毒屬於馬鈴薯Y病毒科(Potyviridae),馬鈴薯Y病毒屬(Potyvirus),使寄主葉片產生嵌紋、變形甚至絲狀化、果實表面出現水浸狀輪斑等病徵。PRSV主要分為P型及W型,P型可感染番木瓜科及葫蘆科,W型只能感染葫蘆科。PRSV-P型目前依照葉片上呈現之病徵又分為嚴重嵌紋(severe mottling, SM)、嚴重嵌紋壞疽(severe mottling with necrosis, SMN)與畸形(deformation, DF)三個主要系統。三系統間具有病理性及分子性之差異,顯示PRSV田間演化情況複雜,目前田間病害以SMN與DF兩系統為主導優勢,兩系統基因序列相似度極高病徵卻截然不同,值得探入探討造成病徵不同之決定因子(symptom determinants)。SMN系統與DF系統全長序列相似度為96.5%,各基因核苷酸序列相似度約為96-99%,靠近5’端的序列相似度較低,如P3基因相似度95.8-96.7%,P1基因低於95%,5’UTR則在93.0-94.2%,本論文將以實驗室SMN與DF兩系統具感染力之全長cDNA選殖株,選擇兩系統間差異性較大之P1基因序列進行重組置換。以精確phusion PCR增幅DF系統之5’端至P1的基因片段,後接SMN的片段,構築成5’-DF(P1)-SMN的重組cDNA選殖株 ; 也以同樣的方法構築5’端至P1為SMN片段,後接DF片段另一重組5’-SMN(P1)-DF選殖株。以胞外轉錄(in vitro transcription)將兩感染性選殖株合成完整的病毒基因體RNA transcripts,機械接種於最感病的木瓜寄主台農二號,發現5’-DF(P1)-SMN重組選殖株的病徵表現以DF為主,5’-SMN(P1)-DF重組選殖株亦然,顯示P1基因對病徵有影響,但不是唯一的病徵決定因子。

Papaya ring spot caused by Papaya ring spot virus (PRSV) , one of the most destructive diseases in papaya. PRSV belonging to the genus Potyvirus, family Potyviridae. PRSV-infected papaya trees show mosaic, distorted and shoestring-like symptoms on the leaves, and sunken ring spots on the fruits. PRSV categorized into 2 types, PRSV-P type and PRSV-W type. The P type infects both papayas and cucurbits whereas the W type only infects cucurbits. Based on the incited symptoms, the PRSV-P type further devided into SM (severe mottling), SMN (severe mottling with necrosis) and DF (deformation) strains. Between these 3 strains have apparently differences on pathological and molecular. This study investigate the genomic variations and find the key genomic areas associated with pathogenicity among different PRSV strains through the research with artificially recombinant infectious clones infecting papaya hosts. Based on the previously constructed infectious clones of PRSV SMN and DF strains, two new recombinant infectious named 5’-DF(P1)-SMN and 5’-SMN(P1)-DF clones were further made. The 5’-DF(P1)-SMN clone has a head of DF (5’UTR and P1 fragment of DF) followed by the SMN fragment, and the 5’-SMN(P1)-DF clone has a head of SMN (5’UTR and P1 fragment of SMN) followed by the DF fragment. The recombinant genomic RNA trascripts were synthesized through in vitro transcription, and they were used to individually inoculate the TN2 papayas. The results showed that symptom express of both two recombinant clones similar to PRSV-DF. It revealed that P1 gene contribute part of pathogenicity, but not the only one gene affect symptom express.

目錄
壹、前言 1
貳、前人研究 3
一、木瓜輪點病之起源與危害 3
二、木瓜輪點病毒分類與分子特性 4
三、木瓜輪點病毒之內含體 4
參、材料與方法 15
一、試驗植物準備 15
二、病毒來源 15
三、人工重組感染性選殖株構築 15
1. PCR (使用phusionTMHigh-Fidelity DNA Polymerase) 15
2. PCR (使用LA TaqTM PCR polymerase) 15
3. 酒精沉澱法 16
4. 接合作用(Ligation) 16
5. 電穿孔 16
6. 篩選並定序確認 16
7. 微量質體純化 17
四、胞外轉錄(in vitro transcription) 17
五、接種試驗 18
六、木瓜輪點病毒之偵測方法 18
1. 病毒核酸萃取 18
2. Two-Step RT-PCR 18
3. One-Step RT-PCR 19
4. PCR產物電泳膠體分析 19
七、菌株繼代培養 21
1. 轉型作用(transformation) 21
陸、參考文獻 33
柒、圖與表 45
捌、附錄 70


表目錄
表一、木瓜輪點病毒重組選殖株(Papaya ringspot virus recombinants)基因核酸片段之構築所用引子對及其基因產物 46
表二、木瓜輪點病毒重組選殖株(Papaya ringspot virus recombinants)基因核酸片段之定序所用引子對及其基因產物 47
表三、以反轉錄聚合酶連鎖反應(RT-PCR)與即時定量反轉錄聚合酶連鎖反應(real-time RT-PCR)偵測木瓜輪點病毒(PRSV)所使用引子及探針 48
表四、病徵整理比較 49
表五、人工構築5’-DF(P1)-SMN、5’-SMN(P1)-DF與DF、SMN之P1核酸和胺基酸序列相似度和歧異度比對 50
表六、接種前5’-DF(P1)-SMN、5’-SMN(P1)-DF與接種後5’-DF(P1)-SMN、5’-SMN(P1)-DF之P1核酸和安基酸序列相似度和歧異度比對 51


圖目錄
圖一、木瓜輪點病毒(PRSV)人工重組P1片段之選殖策略 52
圖二、構築5’-DF(P1)-SMN infectious clone 53
圖三、構築5’-SMN(P1)-DF infectious clone 54
圖四、重組人工質體全長基因體cDNA胞外轉錄後以XbaI酵解為線狀 56
圖五、人工重組後木瓜輪點病毒(PRSV)全長cDNA選殖株之胞外轉錄RNA產物電泳分析 57
圖六、人工重組選殖株DF、5’-DF(P1)-SMN、SMN及5’-SMN(P1)-DF接種後以通用性引子對PRSV-476、DF626、SMN466進行RT-PCR偵測 58
圖十三、台農二號接種5’-SMN(P1)-DF選殖株胞外轉錄產物30天、60天、90天、100天後病徵表現 65
圖十四、5’-SMN(P1)-DF繼代接種在台農二號木瓜上引起之病徵 66
圖十五、台農二號接種DF選殖株胞外轉錄產物30天、60天、90天、100天後病徵表現 67



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