臺灣博碩士論文加值系統

中文摘要
　　台灣瓜類栽培面積廣大且種類繁多，其中以甜瓜及西瓜為大宗。此二作物受矮南瓜黃化病毒 ( Zucchini yellow mosaic virus, ZYMV ) 及木瓜輪點病毒西瓜系統 ( Papaya ringspot virus W type, PRSV-W ) 之危害，造成嚴重損害，由於缺乏抗病材料，傳統方法對於此二病毒的防治迄無良方，本研究乃利用遺傳工程方法構築具 ZYMV及PRSV-W病毒之鞘蛋白轉基因瓜類，預期能得到抗病毒的種原。本實驗以含NPTII和同時帶矮南瓜黃化嵌紋病毒 ( Zucchini yellow mosaic virus, ZYMV ) 及木瓜輪點病毒西瓜型 ( Papaya ringspot virus W type, PRSV-W ) 相連鞘蛋白基因的農桿菌為轉殖媒介，並以網紋洋香瓜商用栽培種的種子進行轉殖，試圖建立一套適合本土洋香瓜栽培品種之基因轉殖與組織培養再生系統。
　　經由建立組織培養叢生苗過程發現，預備試驗中，分別探討添加不同的維他命至基本培養基中，植株產生不正常的癒合組織及嚴重水浸狀現象，將培養基中thiamine HCl成分濃度提高後，確實有效降低水浸狀的現象。使植株生長正常，其最適合的濃度為50 mgl-1。基因轉殖及再生的實驗，以成熟種子子葉為材料，經去殼消毒後，每片子葉切割成四等份，感染農桿菌後，經四天的共同培養後，在含有抗生素的培養基中進行篩選，直到形成擬轉基因芽體後，再移入芽體篩選培養基繼續進行篩選培養。結果顯示，單獨添加BA 0.5 mg l-1的再生培養基再生率為51.7 %，並且以添加抗生素carbenicillin作為篩選，效果最佳，並得到十個以上正常的擬轉基因株系。由PCR放大偵測到病毒鞘蛋白基因及NPTII確實在約1.0 kb的位置有明顯之位帶。並且由南方點漬法發現，共三個轉基因株系( ZW-2、ZW-3及ZW-4 )為2個重複序列數目( copy number )。形成單一芽體後，再進行後續的發根及馴化處理，至溫室中進行溫室抗病評估的分析，期望能成功構築具有抗病性的轉基因網紋洋香瓜。
關鍵字：網紋洋香瓜、組織培養、基因轉殖、再生、農桿菌

ABSTRACT
Zucchini yellow mosaic virus ( ZYMV ) and Type W strain of Papaya ringspot virus ( PRSV-W ) are transmitted by aphids and cause serious economical loss to cucurbit cultivation in Taiwan. The major objective of this study was to establish a better microprogated system and produce coat-protein-gene transgenic plants that are resistant to ZYMV and PRSV-W.
Muskmelon multiple shoots exhibited hyperhydric appearance when cultured in vitro and poor survival rate under acclimatization. In this study, we tried to decrease the hyperhydric rate by adding different concentration of thiamine HCl ( 10 mgl-1,50 mgl-1,100 mgl-1 ) to the MS basal medium. The results indicated that the rate of hyperhydric shoots was reduced when the shoot culture or the medium with 50 mgl-1 thiamine HCl.
In the transformation procedures, seed coats or mature seeds were removed and the seeds was sterilized as explants for transformation . Each fresh cotyledon was soaked on MS solution and cut into 4 pieces using a dull scalpel blade. A fresh overnight bacterial suspension was added 50 μl and light sharked for 10 min. All explants were transferred to the co—culture medium for four days then transplanted to the regeneration medium containing 100 ppm kanamycin and 200 ppm carbenicillin or 200 ppm cefotaxime. The putative transformed buds formed from explants cultured for 4-6 weeks on the selective medium. The best rate of bud formation was 51.7 % on the regeneration medium ( MS basal medium adding 0.5 mg l-1 BA, 200 mgl-1 carbenicillin and 100 mgl-1 kanamycin ). All the buds were removed and transplanted on multiple—elongation medium ( 0.01 mgl-1 NAA & 0.1 mgl-1 BA ) containing 100 mgl-1 kanamycin and 200 mgl-1 carbenicillin. After several times subcultures the buds developed to normal-appearance multiple shoots. We produced more then 10 putative transgenic lines. All the putative transgenic lines were conformed by PCR using specific NPTII or CP gene primer.
In our study, we describe the procedures for regeneration and Agrobacterium—mediated production of transgenic Muskmelon, an economically important cultivar of melon in Taiwan.
Key Words : Muskmelon、tissue culture、transformation、regeneration
、Agrobacterium

目錄
封面內頁
簽名頁
授權書 1.................................... iii
授權書 2.................................... iv
中文摘要.................................... v
英文摘要.................................... vii
誌謝......................................... ix
目錄…………………………………………………… x
圖目錄………………………………………………… xiii
表目錄………………………………………………… xiv
符號說明……………………………………………… xv
第一章　前人研究
1.1 洋香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的發生及特性………… 4
1.4 交互保護策略對抗病毒之研究………………… 5
1.5 洋香瓜基因轉殖之研究………………………… 6
1.6 農桿菌的生理特性及基因轉殖機制…………… 8
第二章　材料和方法
2.1實驗材料……………………………………… 10
2.2實驗方法………………………………………… 11
2.2.1網紋洋香瓜叢生苗組織培養方法之建立 11
2.2.2網紋洋香瓜的再生培養………………… 13
2.2.3網紋洋香瓜基因轉殖-改良式的子葉切
割法……………………………………… 14
2.2.4轉基因株系之分子分析………………… 14
2.2.4.1 植物總 DNA 抽取法…… …… 15
2.2.4.2 聚合酵素連鎖反應… 15
2.2.4.3 南方點漬法 ……… 16
2.2.5轉基因植物之發根及馴化處理………… 18
2.2.6轉基因株系的溫室評估………………… 18
2.2.6.1溫室評估……………………… 19
2.2.6.2自交留種……………………… 20
第三章　結果
3.1 網紋洋香瓜叢生苗組織培養技術建立之探討 21
3.2 不同處理對網紋洋香瓜再生試驗…………… 24
3.3 網紋洋香瓜基因轉殖………………………… 25
3.4 網紋洋香瓜轉基因株系之分子分析………… 26
3.5 轉基因株系溫室抗病評估…………………… 27
第四章　結論…………………………………………………… 29
參考文獻………………………………………………………… 48
附錄一　台灣栽培之甜瓜( Cucumis melo L. )種類………… 57
附錄二　常用的基因轉殖方法………………………………… 58
附錄三　基因轉殖作物………………………………………… 59
附錄四　農桿菌之T-DNA轉殖模式…………………………… 60
附錄五　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……………………………………… 61
附錄六　植物總 DNA抽取流程圖……………………………… 62
附錄七　專一性引子設計序列………………………………… 63
附錄八　南方點漬法( Southern blotting )裝置圖………… 64
附錄九　網紋洋香瓜發根及馴化處理流程圖………………… 65

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