Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36043
標題: 利用 cDNA-Amplified Fragment Length Polymorphism 分析竹嵌紋病毒感染期間菸草基因表現的差異性
Analysis of Differential Gene Expression in Nicotiana benthamiana During Bamboo Mosaic virus Infection Using cDNA-Amplified Fragment Length Polymorphism
作者: 鄭舜方
Cheng, Shun-Fang
關鍵字: cDNA-Amplified Fragment Length Polymorphism
出版社: 生物科技學研究所
摘要: 
植物在自然界中會受到各種不同的病原體侵害,例如病毒、細菌、真菌、無脊椎動物甚至於其他植物的侵襲。植物受到病原體入侵時其基因的表現情形通常會伴隨著有所改變,而植物基因表現情形的改變可能是因病原體入侵的過程所造成的,也有可能是植物的防禦機制反應,使得某些基因的表現量被改變。而在病原體與寄主蛋白的交互作用中,這些表現量被改變的基因可能扮演著重要的角色,本實驗就是要尋找這些表現量有差異的基因,並期能進一步的加以探討其功能。本實驗所選用的病原體研究材料為竹嵌紋病毒(Bamboo mosaic virus, 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絲狀病毒。此病毒在台灣地區對於竹類作物的品質與產量造成極為嚴重的的危害。為了研究植物被此病毒感染時其基因表現的差異的情況,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差異。在實驗中,分別從接種水及接種病毒後的第一、第三、第五及第七天的葉片中萃取total RNAs,然後從這些total RNAs 中進一步純化出mRNAs,並以其為模板來合成雙股的cDNA。 之後即以cDNA-AFLP這個方法來針對植物被病毒感染後,其基因的表現情形做全面系統性的比較與探討。我們將cDNA利用二種限制酵素(TaqI及MseI)加以截切,接上設計好的adapters,依照己知序列的adapters設計primers進行預增殖(pre-amplification)反應。經由八組引子對的增殖結果,我們偵測到六十六個表現量有差異的基因片段,其中六個基因片段經過解序後,並將其之預測蛋白質序列與Genebank的資訊比對後,我們比對到了四個基因,分別為:receptor-like protein kinase,thaumatin-like protein,glutathione S-transferase 及membrane related protein,另外兩個基因並沒有和任何發表之基因有較高的相似度。

Plants are under the constant threat of attack by pathogens and they have evolved mechanisms to counter it. Upon infection by a pathogen, the plant takes counter measures against the pathogen through differential gene expression i.e. by turning certain genes on and certain genes off. This differential gene expression may be either in response to the infection or could be under the influence of the infecting pathogen especially in the case of an obligate intra cellular pathogen like a virus. The products of some of the genes that are turned on may interact with the replication machinery of the virus and influence the process of viral propagation. My interest is to make sense out the patterns of differential gene expression obtained during viral infection, by identifying the genes that are turned on or off in response to the infection of Bamboo mosaic virus (BaMV). BaMV is a filamentous, single stranded positive-sense RNA virus. It can cause severe damage to bamboo plantations upon infection, inflicting huge economic loses. In exploiting the host response that the genes are turned on or off specifically in response to BaMV infection, which in this study is done by cDNA-amplified fragment length polymorphism (cDNA-AFLP). We compare the pattern of expression between mock-inoculated and BaMV-inoculated Nicotiana benthamiana leaves. The total RNAs from the leaves collected at 1, 3, 5, and 7 days post inoculation with or without BaMV were used to purify mRNA. These mRNAs were used as templates to synthesize double-strand cDNA. Then we use cDNA-AFLP technology to study the pattern of gene expression. In this thesis, as a trial to validate the system, eight primer pair combinations were used to analysis differential gene expression. We got 66 differentially expressed genes and six of them were sequenced.
URI: http://hdl.handle.net/11455/36043
Appears in Collections:生物科技學研究所

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