Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35852
標題: 菸草碳酸酐酶與竹嵌紋病毒感染週期之鑑定
Identification of the involvement of carbonic anhydrase of Nicotiana benthamiana in the Bamboo mosaic virus infection cycle
作者: 蔡濬鈺
Tsai, Jun Yuh
關鍵字: 碳酸酐酶竹嵌紋病毒;carbonic anhydrase;基因靜默;Bamboo mosaic virus;gene silencing
出版社: 生物科技學研究所
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摘要: 
Bamboo mosaic virus (BaMV) is a single-stranded, positive-sense RNA virus. In our previous study, we used cDNA-AFLP technique to discover some differentially expressed gene fragments upon BaMV inoculation on Nicotiana benthamiana. These gene fragments may play a vital role in the infection cycles of BaMV and thus enables us to discover the interaction between host factors and pathogens. One of the down-regulated genes, ACAC10-1, which is the partial sequence of carbonic anhydrase gene of N. benthamiana clone 30F62 chloroplast, was then further illustrated. Our first goal is to explore whether ACAC10-1 plays a restricted or enhanced role upon virus inoculation. The result suggested carbonic anhydrase might be required in the virus infection cycle. Our second goal is to identify whether ACAC10-1 is involved in virus replication or movement. After inoculating viral RNA into protoplasts derived from the knockdown plants, this result indicated ACAC10-1 is very likely to be involved in the replication of BaMV. To reveal the replication defect is mainly on plus- or minus-strand RNA synthesis of BaMV, our third goal is using the qRT-PCR to evaluate the accumulation of both plus- and minus-strand of RNAs. We could conclude from the data that the deficiency is possibly on the synthesis of minus-strand RNA. The next aim is to determine whether ACAC10-1 is only involved in infection cycle of this particular virus or not. Preliminary results demonstrated ACAC10-1 might play a virus-specific role upon virus infection. After obtaining the full length of carbonic anhydrase in N. benthamiana (NbCA) which has not yet been published, we fused NbCA with mOrange2 reporter gene to generate NbCA1-orange construct and transiently expressed it by agroinfiltration. Confocal fluorescence microscopy confirmed the fluorescence was observed in chloroplasts. However, transiently expressed NbCA did not lead to considerable increase of the accumulation of virus coat proteins.

竹嵌紋病毒(Bamboo mosaic virus, BaMV)為一彎曲絲狀、單股之正極股RNA病毒。在先前實驗中,我們利用cDNA amplified fragment length polymorphism (cDNA-AFLP) 技術鑑定出一些在竹嵌紋病毒感染後,具有差異性表現的基因片段,而這些片段有可能調節了竹嵌紋病毒在寄主體內的累積,我的實驗目的就是針對其中的一個基因片段進行深入的探討其在竹嵌紋病毒感染週期中所扮演的角色。本次實驗所鑑定的基因片段為ACAC10-1,是屬於煙草植物(Nicotiana benthamiana)內的部分碳酸酐酶(carbonic anhydrase)基因。首先,我們利用病毒誘導基因靜默技術(VIGS)來抑制ACAC10-1在菸草中的表現,發現與控制組相比,竹嵌紋病毒的外鞘蛋白累積量有明顯的下降趨勢,此結果讓我們推論出ACAC10-1在竹嵌紋病毒感染週期中所扮演的角色為幫助病毒增殖,而不是抑制病毒生長。第二個目標則是利用靜默了ACAC10-1的原生質體,進一步釐清ACAC10-1是影響於病毒的複製階段,而不是病毒之移動過程。此外,也利用及即時聚合酶連鎖反應(real-time PCR)的方式,量測出在靜默了ACAC10-1的原生質體中的病毒正負股基因體核酸累積量比例關係與對照組中的累積量相比,推論出ACAC10-1應該是參與了病毒的負股核酸基因體的合成步驟。接著我們也接種了其他兩種病毒,來推敲ACAC10-1是否只專一影響竹嵌紋病毒,結果顯示ACAC10-1只對BaMV有影響。最後,在得到菸草的碳酸酐酶之基因全長後,我們將此全長與mOrange2報導基因融合,再使用agro-infiltration之方法使此融合蛋白在菸草細胞表現。透過共軛焦顯微鏡的觀察,發現此蛋白在菸草細胞內的位置是座落於葉綠體內;且在病毒感染後,植株內的碳酸酐酶位置並沒有明顯改變,同樣是座落於葉綠體內。然而,大量表現此碳酸酐酶再接種竹嵌紋病毒後,發現病毒並沒有明顯增加的趨勢,說明雖然靜默了ACAC10-1會影響病毒複製,但大量表現之卻不會強化病毒以感染寄主。
URI: http://hdl.handle.net/11455/35852
其他識別: U0005-0908201219244400
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