Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90076
標題: The study of the differentially expressed gene ACCT7-1 of Nicotiana benthamiana is involved in Bamboo mosaic virus infection cycle
菸草差異性表現基因ACCT7-1參與竹嵌紋病毒複製之研究
作者: 蔡茜雯
Chien-Wen Tsai
關鍵字: 竹嵌紋病毒;Bamboo mosaic virus
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摘要: 
竹嵌紋病毒(Bamboo mosaic virus, BAMV)屬於彎曲病毒科(Flexiviridae) 中的馬鈴薯病毒屬(Potexvirus),為以單一正股RNA 為遺傳物質的絲狀植物病毒。在病毒複製、移動的過程之中,往往會包含許多宿主基因的參與與調控,為了找到並了解這些宿主基因,本實驗室在先前的研究中利用了cDNA amplified fragment length polymorphism (cDNA-AFLP) 技術去檢視菸草植物(Nicitiana benthamiana),並篩選出九十個在感染竹嵌紋病毒後會產生表現差異的基因,這些基因很有可能跟病毒的增殖相關。本實驗針對其中的五個 cDNA-AFLP 片段進行研究。首先利用病毒誘導基因靜默(virus induced gene silencing; VIGS)技術分別抑制這幾個基因的表現量,觀察是否造成病毒鞘蛋白累積量差異。而這些基因片段當中的ACCT7-1,其所轉錄的蛋白和大豆(Glycine max)的DELLA 蛋白有高度相似性。DELLA 蛋白是一群調控植物生長的蛋白,為植物生長抑制因子,其主要是以抑制植物生長激素:吉貝素(gibberellin; GA) 來調控植物生長,而GA-INSENSITIVE (GAI) 為DELLA蛋白其中的一種。在先前的實驗已發現在植物感染病毒後,ACCT7-1表現量會上升,而當我們以VIGS抑制此基因片段表現之後,病毒累積量也有明顯下降。為了進一步知道ACCT7-1 是影響在病毒的複製或是移動,我們用VIGS 處理菸草的原生質體(protoplast),並接種竹嵌紋病毒之RNA ,發現病毒鞘蛋白表現量和在植物中一樣有下降的趨勢,顯示此基因是影響在病毒的複製。為了能夠進一步了解菸草GAI蛋白質參與竹嵌紋病毒複製的機制,我利用已知的GAI蛋白設計引子,將菸草植物中的GAI基因選殖出來,以便在菸草植物中表現,進而檢視其對竹嵌紋病毒的複製效率是否有增強的作用以及作用之位置。

Bamboo mosaic virus (BaMV) is a single-stranded, positive-sense RNA virus. It belongs to the Potexvirus of Flexiviridae. In general, host genes are participating in various steps of virus infection cycle such as replication, translation, and movement. To identify these host genes, our lab used cDNA amplified fragment length polymorphism (cDNA-AFLP) technique to screen the differentially expressed Nicotiana benthamiana genes after BaMV inoculation. I choose five of these differentially expressed cDNA fragments and test them by using virus induced gene silencing (VIGS) technique to examine whether these genes are involved in the infection of BaMV. One of the fragments, ACCT7-1, encodes a portion of polypeptide with the sequence matched to GA-INSENSITIVE (GAI), a member of the DELLA family, of Glycine max. DELLA is a group of proteins that regulate plant growth by inhibiting the activity of gibberellin (GA), an important plant hormone which induce plant growth. ACCT7-1 was down regulated after BaMV inoculation. The accumulation of BaMV is decreased when the expression of ACCT7-1 was knocked down by VIGS. Further, to clarify whether ACCT7-1 is participating in the replication or movement of BaMV in N. benthamiana, the knockdown protoplasts were inoculated with BaMV. The result showed that the accumulation of BaMV was down regulated as that shown in plants. To gain more information about this gene, I designed primers by a presented GAI gene sequence to clone the full-length of GAI gene, and subcloned it into pEpyon to get the NbGAI1-OFP construct for expressing it in N. benthamiana to examine how this protein is involved in virus replication and its position within the cells.
URI: http://hdl.handle.net/11455/90076
Rights: 同意授權瀏覽/列印電子全文服務,2015-02-07起公開。
Appears in Collections:生物科技學研究所

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