The study of ferredoxin-NADP+ oxidoreductase involved in Bamboo mosaic virus replication in Nicotiana benthamiana

竹嵌紋病毒(Bamboo mosaic virus, BaMV)為一個單股正極核糖核酸病毒，隸屬於Alphaflexiviridae科Potexvirus屬。我們以cDNA-AFLP的技術在菸草植物(Nicotiana benthamiana)中篩選出在竹嵌紋病毒感染後具有差異性表現的宿主基因片段。其中一個基因片段ACAG1，在感染後基因表現下降，在與資料庫進行比對後，發現ACAG1基因與資料庫中的非專一性ferredoxin NADP+ reductase的序列有高度相似性，我們將此基因命名為NbFNR。FNR是一種氧化還原酶，參與了光合作用過程中電子傳遞鏈催化NADP+轉變成NADPH的反應。為了探討NbFNR是否影響竹嵌紋病毒的感染週期，將其基因片段利用病毒引發之基因靜默技術(virus-induced gene silencing，VIGS)使其表現量下降，分別在葉片和原生質體(protoplast)接種病毒後偵測病毒核酸，發現竹嵌紋病毒的蛋白與核酸累積量比對照組有顯著減少。進一步的實驗中我們將NbFNR接上報導基因(Orange fluorescent protein gene)和T7 tag後透過大量表現此基因後也觀察到，竹嵌紋病毒的外鞘蛋白和控制組相比累積量有明顯的上升，同時利用共軛焦顯微鏡觀察NbFNR蛋白在葉片細胞內的位置，發現NbFNR會座落在細胞的葉綠體中，以上這些結果指出了NbFNR在竹嵌紋病毒感染週期中可能扮演幫助的角色。Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to the genus Potexvirus of the family Alphaflexiviridae. We used cDNA-AFLP technique to isolate the host gene fragment which had differential expression after BaMV infection in tobacco plant (Nicotiana benthamiana). One of downregulated gene fragments, ACAG1, is sharing high identity to the non-specific ferredoxin NADP+ reductase sequence in the database; we then designated this gene NbFNR. It is a flavoenzyme that involved in the process of photosynthesis electron transport chain, catalyzes NADP+ into NADPH reaction. In order to investigate whether NbFNR affects the infection cycle of BaMV, we used virus-induced gene silencing (VIGS) technique to reduce the expression level of NbFNR gene in leaves and protoplasts. After BaMV inoculation, the accumulation of BaMV coat protein and viral RNA was significantly lower than that of the control group. Further experiment of transiently expressed NbFNR fused with T7-tag or Orange fluorescent protein (OFP) in plants that could elevate the accumulation of BaMV coat protein. The localization of NbFNR-OFP was observed at chloroplast as expected by confocal microscopy. Overall of these results suggest that NbFNR localized at the chloroplast plays a positive role in BaMV replication.中文摘要(i) Abstract(ii) Introduction(1) Bamboo mosaic virus(1) Ferredoxin-NADP+-Oxidoreductase(2) Aims(3) Materials and Methods(4) Plants growth condition and virus(4) Virus particle purification(4) Viral RNA extraction(4) Virus-induced gene silencing(5) Protoplasts isolation and virus RNA inoculation(5) Western blot analysis(6) Total RNA extraction from leaves(6) Total RNA extraction from protoplasts(7) qRT-PCR(7) Transient expression and localization of NbFNR(8) Northern blot analysis(8) Results(10) Reducing the expression of NbFNR resulting in chlorosis of N. benthamiana and decrease the accumulation of BaMV RNA(10) The accumulation of BaMV was decreased in NbFNR-knockdown protoplasts(11) The accumulation of BaMV coat protein is increased in NbFNR-transiently expressed plants(12) NbFNR is localized in chloroplasts of N. benthamiana(12) Discussion(13) Conclusion(14) Figures(15) Figure 1.The phenotype of NbFNR-knockdown plants and knockdown efficiency.(15) Figure 2.The relative accumulation of BaMV RNA in NbFNR-knockdown plants.(16) Figure 3.The relative accumulation of BaMV coat protein in NbFNR-knockdown protoplasts.(17) Figure 4.The relative accumulation of BaMV RNA in NbFNR-knockdown protoplasts(18) Figure 5. The accumulation of BaMV coat protein in NbFNR-transiently expressed plants.(20) Figure 6. The expression and localization of NbFNR-OFP in N. benthamiana cells(21) Supporting Information(22) References(25