Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31053
標題: 利用蕪菁嵌紋病毒HC-Pro、CIP、NIb和CP基因之高度保留區開發對馬鈴薯Y群病毒具有廣泛抗性之轉基因菸草
Development of transgenic resistance against potyviruses in tobacco plants carrying highly conserved regions of HC-Pro, CIP, NIb and CP genes of Turnip mosaic virus
作者: 吳承祐
Wu, Cheng-Yu
關鍵字: Transgenic plant
轉基因植物
post-transcriptional gene silencing
potyviruses
broad spectrum resistance
後轉錄基因沉寂作用
馬鈴薯Y群病毒
廣泛性抗性
出版社: 植物病理學系所
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摘要: 馬鈴薯Y群病毒屬(Potyvirus屬)在全世界許多重要的經濟作物造成嚴重的危害。為了能夠有效且廣泛性的對抗馬鈴薯Y群病毒所造成的病害,利用抗病毒的轉基因作物,使其明顯地降低在經濟上的損失是最好的方法。本研究利用轉基因技術,將蕪菁嵌紋病毒(Turnip mosaic virus, TuMV) 的HC-Pro、CIP、NIb和CP基因的高保留序列轉殖於菸草上,擬經由後轉錄基因沉寂作用(post-transcriptional gene silencing, PTGS),誘發轉基因菸草產生對馬鈴薯Y群病毒屬病毒之廣泛性抗性。為了研究不同高保留序列之構築所產生之抗性差異性,本實驗轉殖出帶有不同高保留序列所組合成的序列之轉基因菸草。結果顯示,帶有不同高度保留序列組合之轉基因菸草株系對於蕪菁嵌紋病毒的高抗性比例相近,並無明顯差異,並且都能對蕪菁嵌紋病毒產生免疫抗性(immunity) 的能力。進一步探討對蕪菁嵌紋病毒具有免疫抗性的轉基因菸草,對其他馬鈴薯Y群病毒是否也具有抗性,因此選擇與蕪菁嵌紋病毒演化親緣性有不同距離的另外四種馬鈴薯Y群病毒挑戰接種於對蕪菁嵌紋病毒免疫的轉基因菸草,分別為菜豆普通嵌紋病毒(Been common mosaic virus, BCMV)、甘藷潛伏病毒(Sweet potato latent virus, SPLV)、山藥嵌紋病毒(Yam mosaic virus, YMV) 和百香果木質病毒 (Passionfruit woodiness virus, PWV)。其結果指出帶有HC-Pro和NIb高保留序列的轉基因菸草挑戰接種山藥嵌紋病毒、菜豆普通嵌紋病毒以及百香果木質病毒時,能夠就延緩病徵的發展有4-25天的影響。北方墨點分析顯示轉基因菸草中小干擾RNA (small interfering RNA, siRNA) 的累積量與抗性呈正相關。因此在個別的基因的比較下,我們認為帶有HC-Pro和NIb基因高保留區序列的轉基因植物能夠對馬鈴薯Y群病毒具有廣泛性的抗性。此外,病毒在感染帶有多基因的轉基因作物時,其基因突變導致對轉基因具有抗性之機會會遠比在單一轉基因作物上發生的機率低很多,因此我們認為帶有多個病毒高保留基因序列的轉基因作物對病毒防治上具有較好的耐久性。本研究結果提供未來發展對馬鈴薯Y群病毒具有廣泛性抗性的轉基因作物之重要的設計模式與參考。
Many economically important crops are attacked by a number of species of the genus Potyvirus. Appropriate transgenic plants conferring multiple resistance against potyviruses may help reduce the agricultural loses significantly. In this investigation, transgenic Nicotiana benthamiana plants carrying the conserved regions of potyviral HC-Pro, CIP, NIb and CP genes, engineered from Turnip mosaic virus (TuMV), were generated for control of potyviruses by post-transcriptional gene silencing (PTGS) mechanism. To compare the effectiveness of different combinations of the conserved regions for confering resistance against potyviruses, different transgenic lines carrying single, double, triple and tetra combinations of the conserved regions were generated by Agrobaterium-mediated transformation. In a similar proportion, transgenic tobacco lines carrying each construct were able to provide complete resistance of immunity to TuMV infection. To assess the broad-spectrum resistance of the transgenic lines, all TuMV-immune lines were further challenged with four other potyviruses, i.e., Bean common mosaic virus (BCMV), Sweet potato latent virus (SPLV), Yam mosaic virus (YMV) and Passionfruit woodiness virus (PWV), which have different phylogenetic distances from the homologous TuMV. The results showed that TuMV-immune tHC5-16, tNIb7-11, tHC20-13+CIP, tHC+CIP+NIb30-12 and tHC+CIP+NIb+CP32-5 transgenic tobacco lines conferred different levels of resistance in delaying symptom expression (4 - 25) after separate inoculation with YMV, BCMV and PWV. Northern hybridization analysis revealed that the levels of accumulation of siRNA were positively correlated to the levels of resistance of transgenic lines. It is inferred that the multiple combination of the conserved regions has a better effect for resistance durability, since the chances for mutations at multiple genes to overcome PTGS by a potyvirus are much less than mutations at a single gene. Among the four individual single constructs, we conclude that the transgenic lines carrying the conserved regions of HC-Pro or NIb gene have higher capability for conferring broad-spectrum resistance against potyviruses.
URI: http://hdl.handle.net/11455/31053
其他識別: U0005-2208201120241900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2208201120241900
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