Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36100
標題: 利用cDNA-Amplified Fragment Length Polymorphism探討菸草感染竹嵌紋病毒具有差異性表現之基因
cDNA-Amplified Fragment Length Polymorphism Analysis of Differential Gene Expression of Nicotiana benthamiana infected by Bamboo mosaic virus
作者: 陳家怡
Chen, Chia-I
關鍵字: BaMV;竹嵌紋病毒;VIGS;cDNA-AFLP;Differential gene expression;病毒誘導基因靜默
出版社: 生物科技學研究所
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摘要: 
竹嵌紋病毒(Bamboo mosaic virus, BaMV) 為一長絲狀,單一正股核醣核酸病毒,屬於馬鈴薯X 病毒屬(Potexvirus) 。主要宿主為竹,並已對竹相關產物造成嚴重的危害。一般來說植物在感染病毒後,植物細胞內的基因體基因表現情形會改變,有些基因可能被誘導而大量表現,亦可能被抑制而導致基因靜默現象。本研究內容主要是觀察菸草(Nicotiana benthamiana)在被竹嵌紋病毒感染後的基因表現型式。我們利用cDNA amplified length polymorphism (cDNA-AFLP),經增幅選殖以及定序比對後發現其中有三個具有差異性表現的基因。Putative 26S proteasome non-ATPase regulatory subunit 11基因在病毒感染後的第三、五、及七天會有表現量下降的趨勢;而non-phototropic hypocotyl protein-like基因在病毒感染後第三及第五天表現量有上升的趨勢;另有一未知基因在感染病毒後的第一、三、五、及七天會有降低表現量的趨勢。我們設計引子針對這些基因進行real-time PCR來確認這些基因的表現是否與cDNA-AFLP所呈現的結果相符合。另一方面,利用基因靜默實驗來探討non-phototropic hypocotyl like protein基因在病毒感染過程中所可能扮演的角色,結果發現在病毒感染後第五天及第七天時有基因靜默的植物有較高的鞘蛋白累積。

Upon viral infection, plants will, in defense, up- or down-regulate their genes. Since virus is an obligate intracellular parasite, host gene expression may be under the influence of viral infection and as a result, some of the gene products would affect virus replication. BaMV is a flexuous-rod, single-stranded positive-sense RNA virus that belongs to the potexvirus group. It infects bamboo plantations and causes damages that inflict huge economic losses. To completely understand the action of BaMV upon infection, we need to know exactly which of the host genes are up and down regulated specifically in response to virus infection. cDNA-AFLP allows the demonstration of differentially expressed transcripts using PCR. Using cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP), I was able to obtain genes that are differentially expressed in response to BaMV infection in Nicotiana benthamiana. Several differential gene expression patterns were observed using the TaqI-GT and MseI-GA primer combination. An unknown gene, a non-phototropic hypocotyl protein-like gene and a putative 26S proteasome non-ATPase regulatory subunit 11 gene were identified. Gene specific primers were designed to quantify gene expression levels using real-time PCR to confirm the results of cDNA-AFLP analysis. Silencing on a NPH-like gene was carried out to examine the effect of silencing on viral coat protein accumulation. At 5 and 7 days post BaMV inoculation of silencing plant, increased accumulation of coat protein can be observed.
URI: http://hdl.handle.net/11455/36100
其他識別: U0005-0202200716502600
Appears in Collections:生物科技學研究所

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