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標題: The study of ferredoxin-NADP+ oxidoreductase from Nicotiana benthamiana involved in the replication of Bamboo mosaic virus
作者: 邱冠智
Guan-Zhi Chiu
關鍵字: 竹嵌紋病毒複製;鐵氧-NADP+氧化還原酶;replication of Bamboo mosaic virus;ferredoxin-NADP+ oxidoreductase
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竹嵌紋病毒(Bamboo mosaic virus)為一個單股正極核糖核酸病毒,隸屬於Flexiviridae科Potexvirus屬。為了找出與竹嵌紋病毒感染週期的宿主基因,我們以cDNA-AFLP的技術在菸草植物(Nicotiana benthamiana)的葉片中,篩選出在竹嵌紋病毒感染後具有差異性表現的基因片段。其中一個基因片段ACAG1,在竹嵌紋病毒感染後基因表現下降。為了探討ACAG1是否影響竹嵌紋病毒的感染週期,我們將ACAG1基因片段利用病毒引發之基因靜默技術(virus-induced gene silencing,VIGS),使ACAG1的基因表現量下降,發現竹嵌紋病毒的外鞘蛋白在葉子的累積量比對照組有顯著增加。此外,竹嵌紋病毒的外鞘蛋白累積量在ACAG1處理VIGS後的原生質體中,比對照組顯著增加1.4倍,並且竹嵌紋病毒RNA累積量也有上升的趨勢。根據這些實驗結果推測宿主基因ACAG1可能抑制竹嵌紋病毒感染週期中的複製階段。 接著,利用rapid amplification of cDNA ends (RACE)技術獲得ACAG1全長序列,並且將得到的全長序列與轉錄子資料庫進行比對,發現ACAG1基因與資料庫中的非專一性ferredoxin NADP+ reductase的序列吻合,因此我們將此基因命名為NbFNR。 為驗證此基因確實能夠具有抑制竹嵌紋病毒在植物中累積的能力,我們將NbFNR在葉片細胞內進行過量表現並利用共軛焦顯微鏡觀察,發現到NbFNR會座落在細胞的葉綠體中,並且大量表現NbFNR後竹嵌紋病毒的外鞘蛋白累積量明顯降低至76%。這個結果更進一步證實了NbFNR可以抑制竹嵌紋病毒在細胞中的累積。由於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。而在文獻研究中發現,植物在黑暗與光照環境下,葉綠體的基質的酸鹼值會受到影響,於是推測基質內的酸鹼值是否會對竹嵌紋病毒造成影響。因此,將接踵竹嵌紋病毒的植物放置黑暗在中生長,發現到竹嵌紋病毒的RNA累積量卻反而有明顯下降。在未來的實驗中,我將會把FNR中的transit peptide以及NAD的結合位分別進行突變,測試NbFNR的這些區段是否有參與抑制竹嵌紋病毒的感染。

Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to genus Potexvirus of family Flexiviridae. To identify the differentially expressed host genes possibly involved in the infection cycle of BaMV, cDNA-AFLP technique was taken to identify those cDNA fragments after infection in Nicotiana benthamiana leaves. One of the downregulated genes ACAG1 is further characterized. To investigate whether ACAG1 is involved in regulating the infection cycle of BaMV, the ACAG1 cDNA fragment is cloned into Tobacco rattle virus (TRV)-based gene silencing vector and transformed to Agrobacterium tumefaciens. When the gene is knocked down by the silencing vector, the accumulation of BaMV coat protein is increase to approximately 2 folds of that of the control. Furthermore, the accumulation of BaMV coat protein in ACAG1-knockdown protoplasts is approximately 1.4 folds of that in the control protoplasts. These results suggest that the host gene ACAG1 could be involved in the replication step of BaMV infection cycle. In addition, the full-length cDNA of ACAG1 is cloned using rapid amplification of cDNA ends (RACE) technique. The identity of the ACAG1 gene is revealed as the non-specific ferredoxin NADP+ reductase (designated as NbFNR) when the sequence of the full-length cDNA is compared to the transcriptome database. Furthermore, NbFNR is transiently expressed in plants and localized in the chloroplasts, the accumulation of BaMV coat protein is reduced to approximately 76% of that of the control. The results suggest that NbFNR could play an inhibiting role in the infection cycle of BaMV. FNR is an oxidoreductase and functioning in the photosynthetic electron transfer chain that catalyzes the NADP+ and H2O to form NADPH and OH-. Since the plants in the light and dark phase were shown to have different affinity betweenFNR and Tic62/Trol and resulting in alkaline and acidic situation in the stroma, respectively. Thus, as BaMV infected in plants and growth in dark, the relative accumulation of BaMV RNA is reduced to 30% of that of the control. The results indicate that the plants in dark might have effect on BaMV RNA replication. Furthermore, I will construct mutants that remove the transit peptide or diminish the NAD binding site to examine the mechanism of the inhibitory role of FNR in BaMV replication.
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