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標題: 利用cDNA-Amplified Fragment Length Polymorphism 分析菸草於竹嵌紋病毒感染期間基因表現的差異性
cDNA-Amplified Fragment Length Polymorphism Analysis of Differential Gene Expression in Nicotiana benthamiana During Bamboo Mosaic Potexvirus Infection
作者: Pei, Hern
關鍵字: BaMV;竹嵌紋病毒;gene expression;cDNA-AFLP;基因表現
出版社: 生物科技學研究所
化防禦反應。本實驗所選用的病原體研究材料為竹嵌紋病毒(bamboo mosaic potexvirus, BaMV ) ,它屬於馬鈴薯病毒群(potexvirus group) ,為一單股正極性核醣核酸基因體病毒。 此病毒在台灣地區對於竹類植物的品質與產量造成了極為嚴重的危害。為了研究植物被病毒感染時所引發的自然防禦機制與病毒如何利用寄主蛋白來幫助生存,我們利用cDNA-amplified fragment length polymorphism (cDNA-AFLP) 的技術來比較被病毒感染及健康的Nicotiana benthamiana葉片基因表現是否有所差異。在實驗中,首先從被病毒感染的第一天、第三天、第五天以及第七天的葉片上萃取出total RNAs,然後從這些total RNAs中純化出mRNAs,並以其為模板來合成雙股的cDNAs。之後即以cDNA-AFLP的程序來針對被病毒引發的基因表現做系統性的全面檢測。先將cDNA經由MseⅠ及 TaqⅠ限制酵素切割後,接上相對應的adapters,並以adapters及限制酵素切位鄰近的序列設計引子進行預增殖(pre-amplification)反應。接著再利用引子3′端上兩個延伸的核酸組合來設計不同的引子來進行選擇性增殖(selective-amplification)反應,經過DNA序列電泳分析,所得到的結果即為cDNA-AFLP圖譜。在各種不同的primer組合反應中,我們發現了一些可以代表病毒感染,而且無法在健康植物中發現的差異性片段。在將這些片段分別進行定序比對後,結果發現其中之一的片段序列與Arabidopsis thaliana 基因體中的一個microtubule-associated protein (MAP) 相似度很高,推測這可能與感染第三天後病毒需要MAP來幫助移動有關。綜合結果顯示,利用cDNA-AFLP的方法可以全面且快速的觀察植物在病毒感染時的基因差異性表現,而觀察到的差異性片段(transcript-derived fragments, TDF)亦可直接做為基因選殖的來源。

Plants must continuously defend themselves against attack from pathogens, namely viruses, bacteria, fungi, invertebrates, and even other plants. Disease resistance in plants often involves recognition of invading pathogens followed by activation of a defense response. Bamboo mosaic potexvirus (BaMV) is a single-stranded positive-sense RNA virus. It can cause severe damage to bamboo plantations upon infection, inflicting huge economic loses. To elucidate the nature of plant response to infection by BaMV, we compared the cDNA-amplified fragment length polymorphism (cDNA-AFLP) pattern of BaMV- and mock-inoculated Nicotiana benthamiana leaves. The total RNAs from the leaves collected at 1, 3, 5, and 7 days post inoculation with BaMV were used to purify mRNA. These mRNAs were used as templates to synthesize double-stranded cDNAs. cDNA-AFLP analysis can systematically explore the host gene expression in response to BaMV infection. First, the cDNA were digested with two kinds of restriction of MseⅠand TaqⅠ, and ligated with adapters. The primers of pre-amplification were designed with the sequence of adapters and cutting site. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. The result of running DNA sequencing gel was the cDNA-AFLP pattern. Several differential patterns were observed with various primer combinations that could be checked for the sequence to identify their respective genes. One among the various fragments has a sequence similarity to Arabidopsis thaliana microtubule-associated protein (MAP) as detected by BLAST. Results showed that cDNA-AFLP can be used as a powerful tool to screen gene differential expression during virus infection, and gene cloning can directly performed from the differential fragments of transcript-derived fragments (TDF).
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