Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36263
標題: 菸草基因ACCT8-1參與竹嵌紋病毒感染週期的研究
The study of the involvement of the host gene ACCT8-1 from Nicotiana benthamiana in the infection cycle of Bamboo mosaic virus
作者: 曾景漢
Tseng, Ching-Han
關鍵字: Bamboo mosaic virus;竹嵌紋病毒;Cladosporium resistance gene;LRR protein;Cladosporium fulvum抗病基因;LRR 蛋白質
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)是一種隸屬於Flexiviridae科Potexvirus屬的正股RNA病毒,是感染竹子的主要病毒之一,由於病毒的基因體較小,能轉譯出的蛋白有限,無法靠自己完成生活史,其需要仰賴宿主細胞提供蛋白質,協助完成複製、摺疊、移動等生命週期。因此,了解病毒及宿主基因間的互動,有助於我們發展對抗病毒的方法。而本實驗的研究目的,是尋找與病毒有關的宿主蛋白質,藉此了解病毒的繁殖機制。根據前人研究之 cDNA-amplified fragment length polymorphism (cDNA-AFLP)結果,我們挑選了一個宿主基因,其在菸草感染病毒後,表現量會明顯隨著時間增加,將之代稱為ACCT8-1,並取得此基因的片段序列。首先,為了比對出這個基因在宿主內可能的用途,我們使用Rapid Amplification of cDNA End (RACE)的技術,延長這個宿主基因的上下游片段,得到3199nt的序列,經過與National Center for Biotechnology Information (NCBI)資料庫比對後,發現ACCT8-1與Nicotiana tabacum的EILP (elicitor inducible LRR protein) gene 及Cladosporium fulvum抗病基因(Cladosporium resistance gene; cf-2 and cf-5)之序列相似。本研究便是以此基因為研究對象,以菸草脆裂病毒(Tobacco rattle virus)誘導基因靜默(virus induced gene silencing; VIGS) 的方法降低其基因的表現,進而以西方墨點法(western blot)來偵測這個基因對於BaMV在宿主內繁殖的影響,結果發現降低ACCT8-1基因表現量後,病毒鞘蛋白(coat protein)的累積量下降了40% (5dpi),而再以北方墨點法(northern blot)偵測病毒RNA累積量的實驗中,也得到相似的結果,但在馬鈴薯X病毒Potato virus X (PVX)及胡瓜嵌紋病毒Cucumber mosaic virus (CMV)中,並沒有發現病毒鞘蛋白累積量有顯著的差別。由此可發現,靜默ACCT8-1基因後,唯有竹嵌紋病毒的累積量有明顯的減少,再進一步再用西方墨點法(western blot)來偵測原生質體(protoplast)及接種葉其上第三片系統葉的病毒鞘蛋白(coat protein)的累積量,研究病毒繁殖量變少的原因,若原生質體(protoplast)中,病毒的累積量減少,則可能是由於病毒的複製量變少,但若是系統葉中的病毒鞘蛋白累積量下降,則可推測是病毒的移動受到了抑制。結果發現,靜默ACCT8-1基因後,系統葉上的病毒鞘蛋白累積量下降40%,但在原生質體中的累積量則只下降10%,由結果得知抑制此基因後,只會導致系統葉中病毒鞘蛋白的累積量明顯下降,因此推斷ACCT8-1基因可能主要是影響病毒的移動能力,因而在抑制其基因表現時,也同時影響了病毒的移動。而且在竹嵌紋病毒感染後觀察病斑的實驗中,也發現基因靜默的植物葉片病斑較小,由此更進一步支持以上的推論。

Bamboo mosaic virus (BaMV), belongs to genus Potexvirus of the family Flexiviridae, is a positive-sense RNA virus. Since the virus can not complete its infection cycle by itself, it needs host factors in different stages, such as RNA replication, folding, and spreading. Therefore, studying the interaction between virus and its host is important and useful in developing antivirus strategy. The aim of this study is identifying the host proteins that involve in the infection of BaMV. According to the previous study, many of differentially expressed genes from BaMV infected Nicotiana benthamiana plants were identified using cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. Then I focus on one of the upregulated cDNA fragments, ACCT8-1. Rapid amplification of cDNA ends (RACE) technique is used to extend the 5' and 3'-end of the cDNA fragments to 3199 nucleotides in length. The sequence the hypothetic protein derived from the coding region is blasted with the database in National Center for Biotechnology Information. ACCT8-1 is an ortholog of N. tabacum EILP (elicitor inducible LRR protein) gene and Cladosporium fulvum resistance gene (cf-2 and cf-5). The expression level of ACCT8-1 is knocked down by Tobacco rattle virus (TRV)-based silencing system to inspect the coat protein accumulation after virus inoculation. Results indicate that the coat protein accumulation is reduced about 40% on the knockdown plants to that of control plants. The accumulation levels of viral RNA analyzed in Northern blot also show similar reduction in knockdown plants. However, the coat protein accumulation levels of Potato Virus X (PVX) and Cucumber mosaic virus (CMV) show no significant difference in ACCT8-1 knockdown and control plants. Further, I also find that the coat protein accumulation has no significant difference and a 40% reduction in the protoplasts and systemic levels derived from the knockdown plants, respectively, compared to those of control plants. Finally, I also find that the lesion sizes in the knockdown plants are smaller than those observed in control plants. Overall of these results suggest that ACCT8-1 may involve in viral movements.
URI: http://hdl.handle.net/11455/36263
其他識別: U0005-2701201105564900
Appears in Collections:生物科技學研究所

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