臺灣博碩士論文加值系統

台灣全年氣候適合瓜類栽植，栽培面積廣大且種類繁多，其中以西瓜和甜瓜為大宗。病毒危害目前仍無任何化學藥劑可防治
，因此常造成瓜類嚴重的經濟損失，其中以西瓜銀斑病毒 (Watermelon silver mottle virus ; WSMoV)、矮南瓜黃化嵌紋病毒(Zucchini yellow mosaic virus ; ZYMV)及木瓜輪點病毒西瓜系統(Papaya ringspot virus Type-W ; PRSV-W)為危害西瓜最嚴重之病毒種類。本研究之前已構築出11個具有ZYMV-PRSV-W複合鞘蛋白之轉基因株系，本研究主要是將這些轉基因西瓜株系，作進一步之溫室接種評估及相關之分子分析。由結果發現line 9及line 10在接種後，利用ELISA、western blotting、RT-PCR及northern blotting分析中，均偵測不到ZYMV及PRSV-W兩病毒的存在。因此，將line 9及line 10歸類為免疫性(immunity)。line 1在複合接種後，外觀上沒有病徵，但卻可測到PRSV-W。所以，將line 1歸類為具有高度抗性(High Resistance, HR)。而line 7有82 %具有ZYMV之抗性，但不抗PRSV-W。本研究發現，所有轉基因株系在未接種前均測不到transgene RNA表現。因而推測，其抗病機制應為RNA-mediated gene silencing之方式。但這些均不表現transgene RNA之株系，卻表現出不同程度抗性(從免疫到完全不抗均有)，因此，RNA-mediated機制仍然還有很多探討之空間。

Watermelon is an economically important crop of the tropics and subtropics. Virus disease often causes serious economic loss of watermelon and there is still no chemical to control the virus disease. Watermelon silver mottle virus (WSMoV), Zuchini yellow mosaic virus (ZYMV) and Papaya ringspot virus Type-W (PRSV-W) are the most hazardous species among all kinds of viruses infected in watermelon. Transgenic watermelon lines carrying ZYMV and PRSV-W coat protein(CP) double fusion gene were previously generated in our laboratory. Therefore, this study was discussed to evaluate resistance of independent transgenic watermelon lines against ZYMV and PRSV-W under greenhouse conditions. Line 9 and line 10 showed immunity against ZYMV and PRSV-W after challenge inoculation. No virus was detected by indirect ELISA, western blotting, RT-PCR, and northern blotting in the immunity transgenic watermelon lines. Line 1 was highly resistant to ZYMV and PRSV-W 30 days postinoculation, showing no symptom. The expression of the transgene in the all transgenic lines was not detected by northern blotting before challenged inoculation. For this reason we consider that the virus resistance is RNA-mediated.

封面內頁
簽名頁
授權書 iii
中文摘要 iv
英文摘要 v
誌謝 vi
目錄 vii
圖目錄 x
表目錄 xi
附錄 xii
符號說明 xiii

1. 前言 1
1.1 西瓜的特性及所面臨的問題 1
1.2 矮南瓜黃化嵌紋病毒之發生及特性 2
1.3 木瓜輪點病毒西瓜系統之發生及特性 4
1.4 交互保護策略對抗病毒之研究 6
1.5 西瓜基因轉殖之研究 8
2. 材料和方法 10
2.1 實驗材料 10
2.1.1 研究材料 10
2.1.2 供轉殖之基因載體的構築 10
2.1.3 基本培養基 10
2.1.4 生長素(如α-Naphthalene-acetic acid, NAA ; 1indole-3-butyric 1acid, IBA)母液之配製 11
2.1.5 細胞分裂素(如6-benzylaminopurine, BA)母液
之配製 11
2.1.6 抗生素母液之配製 11
2.2 實驗方法 11
2.2.1轉基因株系之分子分析 11
2.2.1.1植物總 DNA 抽取法 12
2.2.1.2聚合酶鏈鎖反應 13
2.2.1.3南方點漬法 13
2.2.2轉基因植物之溫室抗病評估及分析 15
2.2.2.1轉基因植物之發根及馴化處理 15
2.2.2.2轉基因株系的溫室抗病評估 16
2.2.2.3間接-酵素連結免疫分析 17
2.2.2.4蛋白質膠體電泳及西方點漬法 17
2.2.2.4.1 蛋白質膠體電泳 18
2.2.2.4.2 西方點漬分析法 19
2.2.2.5植物總 RNA 抽取法 20
2.2.2.6反轉錄酶-聚合酶鏈鎖反應 20
2.2.2.7北方點漬分析法 21
3. 結果 23
3.1 轉基因西瓜株系之分子分析 23
3.2轉基因西瓜株系之南方點漬法分析 23
3.3轉基因西瓜株系之溫室抗病評估 23
3.3.1 轉基因西瓜株系之個別接種試驗 24
3.3.2 轉基因西瓜株系之複合接種試驗 25
3.4轉基因西瓜株系之間接-酵素連結免疫分析之偵測 26
3.5轉基因西瓜株系之西方點漬法分析 27
3.6轉基因西瓜株系之RT-PCR 27
3.7轉基因西瓜株系之北方點漬法分析 28
4. 結論 29
參考文獻 47













圖目錄

圖1. 矮南瓜黃化嵌紋病毒及木瓜輪點病毒西瓜系統鞘蛋白
轉基因西瓜進行聚合酶鏈鎖反應 35
圖2. 南方點漬法分析，矮南瓜黃化嵌紋病毒及木瓜輪點病
毒西瓜系統鞘蛋白轉基因西瓜以nptII質體DNA為探
針，偵測外來基因併入植物基因組之情形 36
圖3. 轉基因西瓜株系於溫室接種矮南瓜黃化嵌紋病毒 37
圖4. 轉基因西瓜株系於溫室接種木瓜輪點病毒西瓜系統 38
圖5. 轉基因西瓜株系於溫室複合接種矮南瓜黃化嵌紋病毒
及木瓜輪點病毒西瓜系統 39
圖6. 轉基因西瓜株系進行複合接種矮南瓜黃化嵌紋病毒及
木瓜輪點病毒西瓜系統在不同時期之Indirect-ELISA
分析 40
圖7. 轉基因西瓜株系進行複合接種矮南瓜黃化嵌紋病毒及
木瓜輪點病毒西瓜系統，在接種後的第15天，以
western blot偵測病毒在轉基西瓜株系之表現情形 41
圖8. 轉基因西瓜株系進行複合接種矮南瓜黃化嵌紋病毒及
木瓜輪點病毒西瓜系統，在接種後的第15天，以
RT-PCR偵測病毒在轉基西瓜株系之表現情形 42
圖9. 轉基因西瓜株系以northern blotting進行偵測未接種及
複合接種矮南瓜黃化嵌紋病毒及木瓜輪點病毒西瓜系
統，觀察轉基因株系之mRNA及病毒在轉基西瓜株
系之表現情形 43

表目錄

表1. 轉基因西瓜株系進行溫室接種矮南瓜黃化嵌紋病毒，
十五天後轉基因植物葉片之發病情形 44
表2. 轉基因西瓜株系進行溫室接種木瓜輪點病毒西瓜系統
，十五天後轉基因植物葉片之發病情形 45
表3. 轉基因西瓜株系進行溫室複合接種矮南瓜黃化嵌紋病
毒及木瓜輪點病毒西瓜系統，十五天後轉基因植物葉
片之發病情形 46















附錄

圖10. 矮南瓜黃化嵌紋病毒及木瓜輪點病毒西瓜系統鞘蛋
白轉基因之構築 54
圖11. 矮南瓜黃化嵌紋病毒及木瓜輪點病毒西瓜系統鞘蛋
白轉基因專一性引子設計序列 55
圖12. 南方點漬法(Southern blotting)裝置圖 56

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