Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90062
標題: The study of the relationship between Bamboo mosaic virus ?infection and its downregulated host gene NbTFIISL from Nicotiana benthamiana
竹嵌紋病毒感染後表現量下降之菸草基因NbTFIISL與病毒感染之相關性研究
作者: 黃瀞瑩
Ching-Ing Huang
關鍵字: 竹嵌紋病毒;宿主差異性表現基因;Bamboo mosaic virus;differentially expressed host gene
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摘要: 
中文摘要
竹嵌紋病毒(Bamboo mosaic virus, BaMV)為單一正股RNA病毒,隸屬於Flexiviridae科Potexvirus屬,為了解此病毒的感染與繁殖機制,本實驗室之前利用cDNA- amplified fragment length polymorphism (cDNA-AFLP)的技術找出90個在竹嵌紋病毒感染後具差異性表現的菸草(Nicotiana benthamiana)基因片段,其中一個表現量下降且經過基因庫比對無相似序列的基因片段ACAC5-2,在經過病毒誘導性基因靜默(virus-induced gene silencing, VIGS)抑制其在菸草細胞內的表現後,接種竹嵌紋病毒,在接種病毒的葉片偵測出其病毒鞘蛋白累積量與對照組相比下降了約百分之四十;另外在基因靜默的原生質體(protoplast)內的病毒鞘蛋白累積量不論在感染竹嵌紋病毒24及48小時後都與對照組沒有顯著性的差異,因此初步推論此基因(ACAC5-2)可能參與了竹嵌紋病毒細胞間的移動。為了進一步針對此基因的功能性分析,以Rapid Amplification of cDNA End (RACE)技術延長了ACAC5-2上下游序列並在菸草的轉錄基因庫中找到可以轉錄出992個胺基酸可能的全長cDNA,另外,利用網路的ScanProsite tool搜尋出在胺基酸序列75~153的位置可能為具有蛋白質結合功能的TFIIS N-terminal domain,在許多幫助延長的轉錄因子例如Transcription Factor IIS、Med26、Elongin A等等都含有這個conserved domain,故命名此基因為NbTFIISL。最後,將這個基因的open reading frame與報導基因mOrange2做結合,利用agro-infiltration表現在菸草葉片上。表現1.5天後,可以透過共軛焦顯微鏡觀察到螢光融合蛋白位在細胞核內。除了確認這個蛋白的功能外,進一步釐清其蛋白與病毒之間的關係是之後研究的主要方向。
中文摘要
竹嵌紋病毒(Bamboo mosaic virus, BaMV)為單一正股RNA病毒,隸屬於Flexiviridae科Potexvirus屬,為了解此病毒的感染與繁殖機制,本實驗室之前利用cDNA- amplified fragment length polymorphism (cDNA-AFLP)的技術找出90個在竹嵌紋病毒感染後具差異性表現的菸草(Nicotiana benthamiana)基因片段,其中一個表現量下降且經過基因庫比對無相似序列的基因片段ACAC5-2,在經過病毒誘導性基因靜默(virus-induced gene silencing, VIGS)抑制其在菸草細胞內的表現後,接種竹嵌紋病毒,在接種病毒的葉片偵測出其病毒鞘蛋白累積量與對照組相比下降了約百分之四十;另外在基因靜默的原生質體(protoplast)內的病毒鞘蛋白累積量不論在感染竹嵌紋病毒24及48小時後都與對照組沒有顯著性的差異,因此初步推論此基因(ACAC5-2)可能參與了竹嵌紋病毒細胞間的移動。為了進一步針對此基因的功能性分析,以Rapid Amplification of cDNA End (RACE)技術延長了ACAC5-2上下游序列並在菸草的轉錄基因庫中找到可以轉錄出992個胺基酸可能的全長cDNA,另外,利用網路的ScanProsite tool搜尋出在胺基酸序列75~153的位置可能為具有蛋白質結合功能的TFIIS N-terminal domain,在許多幫助延長的轉錄因子例如Transcription Factor IIS、Med26、Elongin A等等都含有這個conserved domain,故命名此基因為NbTFIISL。最後,將這個基因的open reading frame與報導基因mOrange2做結合,利用agro-infiltration表現在菸草葉片上。表現1.5天後,可以透過共軛焦顯微鏡觀察到螢光融合蛋白位在細胞核內。除了確認這個蛋白的功能外,進一步釐清其蛋白與病毒之間的關係是之後研究的主要方向。

Abstract
Bamboo mosaic virus (BaMV) belongs to the Potexvirus genus of Flexiviridae family and has a single-stranded positive-strand RNA genome. To understand the infection mechanism BaMV, our lab has selectively detected and isolated 90 differentially expressed genes from Nicotiana benthamiana post BaMV infection using cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique. In my study, one of the downregulated cDNA fragments, ACAC5-2, has no significant match to any known sequence in the databases. The relative coat protein accumulation level of BaMV in inoculated leaves of ACAC5-2 knockdown N. benthamiana plants declined to about 60% that of the control. Furthermore, the coat protein of BaMV in the ACAC5-2 knockdown protoplasts does not show significant change compared to that of the control protoplasts at 24 and 48 hrs post inoculation. These results suggest that the downregulated gene ACAC5-2 could be involved in the cell to cell movement of BaMV. Moreover, The full-length cDNA of ACAC5-2 from N. benthamiana transcriptome database was obtained after up- and down- stream extension by the Rapid Amplification of cDNA Ends (RACE) technique. It could be encoded a 992-amino acid polypeptide comprising a TFIIS N-terminal domain, a conserved protein binding domain found in Transcription Factor IIS, Med26, Elongin A and other transcription elongation factors located at the N-terminal region of this polypeptide. Therefore, this gene was designated NbTFIISL. We further fused this gene with mOrange2 to generate pEyon-NbTFIISL-OFP construct which then transiently expressed by agro-infiltration. The fluorescent signal predominantly located at the nucleus was observed under confocal microscopy. The future work will be focusing on the relationship between this protein and BaMV infection.
URI: http://hdl.handle.net/11455/90062
Rights: 同意授權瀏覽/列印電子全文服務,2015-08-31起公開。
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