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研究生:陳俊麟
研究生(外文):Chung-Lin Chen
論文名稱:具感染力之番茄黃化捲葉病毒之構築與含其複製酶轉基因煙草之抗病分析
論文名稱(外文):The Construction of Infectious Tomato Yellow Leaf Curl Virus(TYLCV) and the Analysis of Resistance on Transgenic Tobacco with TYLCV Replicase Gene
指導教授:江主惠
指導教授(外文):chu-hui chiang
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:2007
畢業學年度:95
語文別:中文
論文頁數:87
中文關鍵詞:番茄黃化捲葉病毒基因轉殖農桿菌聚合酶連鎖反應
外文關鍵詞:Tomato yellow leaf curl virus(TYLCV)transformationAgrobacterium tumefacienspolymerase chain reaction
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番茄黃化捲葉病毒(Tomato yellow leaf curl virus;TYLCV)屬於雙生科(Geminiviridae)中的Begomovirus屬,基因體包含單一種環狀DNA核酸(monopartite genomes),由銀葉粉蝨(Besisia argentifoli)傳播,對番茄收成造成嚴重之危害。本實驗利用病原誘導抗病機制(parasite-derived resistance, PDR)以台灣品系之TYLCV的部分複製蛋白酶基因(C1),依不同排列方式,分別構築正股(sense)、反意股(antisense)及兩段反意股相連(double antisenses)之轉基因質體,再以農桿菌方式轉殖到植株上,並挑戰接種含全長度TYLCV質體之農桿菌。本研究先以煙草為模式,進行抗病分析,以作為將來轉殖到番茄的參考。實驗首先在彰化縣,包括溪湖鎮、永靖鄉、大村鄉及員林鎮八個番茄園區,採集疑似受病毒感染之番茄葉片,再以聚合酶連鎖反應(polymerase chain reaction;PCR)針對病毒之複製酶基因(C1),進行番茄黃化捲葉病毒之偵測,在溪湖鎮40個樣本中,共偵測到5個染病材料,其罹病率為12.5%;在永靖鄉、大村鄉及員林鎮各採集8個樣本,亦各偵測到1個染病植株,罹病率為12.5%。將針對TYLCV的PCR產物進行選殖與解序後,發現無論是C1基因或是全長度基因體,溪湖品系的TYLCV與泰國品系TYLCV最為相近,可達98%。所轉殖含C1基因的擬轉基因煙草,則先利用針對病毒C1基因及NPTII專一性引子進行PCR增幅,可獲得預期DNA片段,證明轉基因確實有送入煙草中,總共每種構築分別得10-15個擬轉基因煙草品系。由於TYLCV無法以機械接種方式感染植物,因此為了進行轉基因煙草的抗病分析,本實驗另外再構築具感染力的TYLCV質體。首先以溪湖採集之TYLCV為材料,以PCR分別放大三段DNA片段,涵蓋全長度病毒基因體,以適當酵素進行剪接後,得到一個含病毒全長度基因體1.27倍TYLCV質體(TI0380AE5),其在5’端多出346核苷酸,在3’端多出416核苷酸,此質體藉由農桿菌,接種到非轉基因煙草及番茄後,取接種植物之上位葉進行PCR及解序,確定此病毒質體(TI0380AE5)確實具有感染力,比正常感染TYLCV的煙草病徵較弱。此具感染力的TYLCV質體即可用來挑戰接種於轉基因煙草。由本實驗在煙草上的研究經驗,將可應用到未來番茄的轉殖上,以達抗番茄黃化捲葉病毒之目的。
Tomato yellow leaf curl virus(TYLCV) is a member of the Begomovirus in the Geminiviridae family. TYLCV contains a monopartite genome and is transmitted by whitefly Bemisia argentifoli and results in severe losses in tomato productions. In this study, naturally infected tomato samples were collected from eight tomato fields in Changhua County and polymerase chain reaction (PCR) were used for the detection of TYLCV using primers pair that is specific to TYLCV genome. Five in the forty samples from Sihu and one in eight samples collected from Yongjin, Dacun, and Yuanlin, respectively, showed positive in PCR analyses. The average infection rate is 12.5%. The PCR products were cloned and sequenced. Sequences alignment with the C1 gene and the full-length genome revealed that TYLCV Sihu isolate is most closely related to a TYLCV Thailand strain, with the nucleotide sequence identity of 98%. By the concept of parasite-derived resistance (PDR), a part of TYLCV replicase gene (C1) was used to engineer into three different transgene constructs for plant transformation, which include the TYLCV C1gene arranged in a sense, antisense and double antisenses manners. These three constructs were transformated into tobacoo (Nicotiana benthamiana) by Agrobacterium tumefaciens. The putative transgenic tobacco plants were selected in Kanamycin medium and confirmed by PCR with the C1 and NPTII specific primer pairs. Ten to fifteen putative transgenic lines were obtained from the transformed of each sense, antisense, and double antisenses constructs. Since it was not possible to analyses the resistant ability of the TYLCV transgenic tobacco by mechanical inoculation of natural TYLCV. An infectious TYLCV clones was constructed. Three overlapping DNA fragment that covered the full-length of TYLCV genome of Sihu isolate were obtained from PCR amplification. The TYLCV clone contains approximately 3.5 Kb that is about 1.27 times longer than virus genome unit size were inoculated into non-transgenic tobacco by argoinoculation. The mild mosaic and less leaf curve symptom were observed 14 days post inoculation. The ability of the infectivity in the inoculated plants were further confirmed by PCR amplification and sequencing. The TYLCV infectious clone was then used as a virus source for challenge into transgenic tobacco and the resistant ability of the transgenic is under investigation. The study of the resistant ability of TYLCV transgenic tobacco can provide the information for the engineering of TYLCV transgenic tomato in the future
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
符號說明 xiv

1. 前言 1
1.1 雙生型病毒科(Geminiviridae)的研究 1
1.2 番茄黃化捲葉病毒之發生 6
1.3 利用轉基因方式的抗病策略 9
1.4 以轉基因方式產生抗Begomovirus病毒屬的研究 11
2. 材料和方法 16
2.1 實驗材料 16
2.2 實驗方法 18
2.2.1 番茄黃化捲葉病毒田間之偵測 18
2.2.2 番茄黃化捲葉病毒引子設計 18
2.2.3 植物總核苷酸(Total DNA)之抽取 19
2.2.4 聚合酶連鎖反應偵測田間番茄黃化捲葉病毒基因 20
2.2.5 E. coli勝任細胞的製備 20
2.2.6 DNA 片段的回收及純化 21
2.2.7 病毒DNA之選殖及解序 21
2.3 煙草轉殖系統之建立 22
2.3.1 轉基因載體之構築 22
2.3.2 構築之質體送入農桿菌 24
2.3.3 煙草基因轉殖 25
2.3.4 擬轉基因煙草之分析 26
2.3.4.1 擬轉基因煙草之總DNA萃取 26
2.3.4.2 以PCR偵測轉基因 26
2.3.5 轉基因煙草之發根及馴化 27
2.4 具感染力番茄黃化捲葉病毒質體之構築 27
2.4.1 番茄黃化捲葉病毒之來源 27
2.4.2 使用之引子 27
2.4.3 全長度基因體之序列比對與演化樹之分析 28
2.4.4 番茄黃化捲葉病毒全長度基因體構築 28
2.4.5 以三溫暖法將質體送入農桿菌 29
2.4.6 接種非轉基因之植物 29
2.4.6.1 非轉基因煙草及番茄(春喜品種) 30
2.4.6.2 農桿菌之培養 30
2.4.6.3 農桿菌接種方式 30
2.4.6.4 病徵觀察 30
2.4.6.5 以聚合酶連鎖反應偵測病毒之感染 31
2.5 轉基因植物之抗病分析 31
2.5.1 轉基因煙草之來源 31
2.5.2 病徵觀察 31
2.5.3 聚合酶連鎖反應偵測其是否感染 31
3. 結果 33
3.1 番茄黃化捲葉病毒之田間偵測 33
3.2 TYLCV基因體之解序、核苷酸比對及演化親緣性分析 33
3.3 植物轉基因之構築 34
3.3.1 正股、反意股及兩段反意股連接在表現載體(pEPJ86)之構築 34
3.3.2 正股、反意股及兩段反意股連接在轉殖載體(pGA482G)之構築 35
3.4 將植物轉殖載體送至農桿菌 36
3.5 煙草轉殖 37
3.6 構築全長度番茄黃化捲葉病毒質體 38
3.7 將含病毒全長度基因體之載體送至農桿菌 39
3.8 構築全長度番茄黃化捲葉病毒質體之感染力分析 39
4. 結論 41
參考文獻 58
附錄 68



圖目錄

圖1. 從彰化縣番茄園各地田間採集疑似感染番茄黃化捲葉病毒之葉片,以TY44及TY43引子進行PCR偵測後之電泳圖 45
圖2. 溪湖品系之番茄黃化捲葉病毒全長度序列與18個TYLCV及一種同屬不同種之AYVV進行親緣性之比對 46
圖3. 番茄黃化捲葉病毒之複製蛋白酶基因送到表現載體pEPJ86及轉殖載體 pGA482G後之簡易構築圖 47
圖4. 構築在表現載體(pEPJ86)及轉殖載體(pGA482G)上轉基因質體,分別以限制酵素HindIII作用後之電泳圖 48
圖5. TYLCV C1的部分基因在植物轉殖載體(pGA482G)之構築圖 49
圖6. 轉基因煙草之組織培養 50
圖7. 含不同構築之擬轉基因植物取其總DNA再以適當引子進行PCR偵測後之電泳圖 51
圖8. 構築全長度含重複序列的TYLCV質體之構築及確認圖 52
圖9. 利用農桿菌感染方式將含全長度TYLCV病毒基因體之載體(TIO380AE)及對照組(不含Ti載體的農桿菌),接種於非轉基因煙草及番茄植物 53

表目錄

表1. 構築含TYLCV replicase ( C1gene ) 之轉基因質體及含全長度具有感染力病毒質體所使用之引子 54
表2. 來自溪湖之番茄黃化捲葉病毒分別與18種TYLCV及一種AYVV序列進行病毒複製酶基因(C1)及病毒全長度基因體序列的相同度比對 55
表3. 擬轉基因煙草培植體以抗生素(Kanamycine)及聚合酶連鎖反應(PCR)偵測 56
表4. 轉基因煙草接種具感染力之infectious clone (TI0380AE5),接種後其發病數目 57
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