Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36850
標題: 抗胡瓜嵌紋、胡瓜綠斑嵌紋與西瓜嵌紋病毒轉基因西瓜之研發
Development of transgenic watermelon plants with multiple resistance to Cucumber mosaic-、Cucumber green mottle mosaic- and Watermelon mosaic viruses
作者: 何琇銀
Ho, Hsiu-Yin
關鍵字: transgenic watermelon
轉基因西瓜
Cucumber mosaic virus
Cucumber green mottle mosaic virus
Watermelon mosaic virus
胡瓜嵌紋病毒
胡瓜綠斑嵌紋病毒
西瓜嵌紋病毒
出版社: 農藝學系所
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摘要: 西瓜是台灣重要的經濟作物,但在栽培期間易受到多種植物病毒的侵害。在先前的研究中顯示當轉基因為silencer DNA連接多個病毒基因片段時可以藉由轉錄後基因沉寂機制提供對多種病毒的抗性,在本研究中使用相似的策略,藉由基因沉寂機制發展出可抗多種病毒的的轉基因西瓜,在本研究中將帶有西瓜銀斑病毒 (Watermelon silver mottle virus, WSMoV) 部份N基因做為silencer DNA其後連接胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV)、胡瓜綠斑嵌紋病毒 (Cucumber green mottle mosaic virus, CGMMV) 及西瓜嵌紋病毒 (Watermelon mosaic virus, WMV) 部份鞘蛋白基因的表現卡匣構築於篩選標示基因與目標基因位於同一T-DNA或不同T-DNA的轉殖載體上,藉由農桿菌轉殖法進行西瓜基因轉殖,利用PCR檢測篩選標示基因,共獲得25個可增幅出篩選標示基因專一片段的基因株系,並利用南方雜合法確認目標基因嵌入西瓜基因組中不同的位置。所獲得的轉基因西瓜分別以CMV、CGMMV、WMV或混合三種病毒進行挑戰接種,評估其對於病毒的抗性,總共25個株系進行接種測試,其中有三個株系顯現出對CGMMV及WMV的抗性,這三個對CGMMV及WMV的抗病株系中有兩個對於CMV及混合接種也具有抗性。利用北方雜合法分析轉基因西瓜植株,可以觀察到抗病表現與低目標基因RNA轉錄累計是有相關的。我們的結果顯示當轉基因西瓜帶有不同的病毒鞘蛋白基因片段可以提供對多種病毒的抗性。目前已獲得由R0自交而得的R1種子,將評估R1植株對於CMV、CGMMV、WMV及WSMoV的廣泛抗性。
Watermelon is an important commercial crop in Taiwan and is prone to attack by several plant viruses. Previous studies have shown that transgenic plants with multiple viral gene fragments linked to an universal silencer DNA can provide resistance to multiple virus via posttranscriptional gene silencing (PTGS). In this study, similar approach was used to develop transgenic watermelon plants resistant to three different viruses via gene silencing. Transformation constructs with selection marker gene and targer gene either in the same T-DNA or in different T-DNAs containing a silencer DNA from the partial N gene of Watermelon silver mottle virus (WSMoV) fused to the partial coat protein genes of Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Watermelon mosaic virus (WMV) were generated and used to transform watermelon plant via Agrobacterium-mediated transformation. A total of twenty-five trnasgenic lines were generated and confirmed by PCR amplification of selection marker gene. Southern blot was used for analyzing the copy number of the target gene inserted the genome of each transgenic watermelon plant. Transgenic watermelon plants were challenged with CMV, CGMMV, WMV or a mixture of these three viruses to evaluate the multiple virus resistance. Among the twenty-five tested lines, three lines showed resistant to CGMMV and WMV. Among these three resistant lines, two were also resistant to CMV and mixed inoculation of three viruses. The result of northern blotting of transgenic watermelon plants showed a correlation between the resistant phenotype and low accumulation level of target gene RNA transcripts. Our preliminary results indicate that transgenic watermelon carrying the fusion of different viral CP gene fragments can provide multiple virus resistance. R1 seeds from the self-pollinated resistant R0 plants were obtained and will be used to evaluate the broad-spectrum resistance to CMV, CGMMV, WMV and WSMoV.
URI: http://hdl.handle.net/11455/36850
其他識別: U0005-2208200700270800
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2208200700270800
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