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標題: 菸草基因ACTG7-1於於竹嵌紋病毒之感染週期中參與的機制
The study of the ACTG7-1 from Nicotiana benthamiana involving in the replication of Bamboo mosaic virus
作者: 王筱禎
Wang, Hsiao-Chen
關鍵字: 竹嵌紋病毒;ethylene;乙烯;BaMV
出版社: 生物科技學研究所
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竹嵌紋病毒(Bamboo mosaic virus; BaMV)為正股RNA病毒,隸屬於Flexiviridae科Potexvirus屬。由於病毒基因體通常較小,因此能夠生合成的蛋白質數量受到限制,所以病毒必需仰賴宿主因子的幫助,才能完成整個生命週期。因此為了找出可能參與病毒感染週期的宿主基因,在先前研究中,本實驗室利用cDNA Amplified fragment length polymorphism (cDNA-AFLP) 技術來篩選在竹嵌紋病毒感染菸草(Nicotiana benthamiana)後表現量有差異的基因片段。其中一個基因片段ACTG7-1,其轉譯出的蛋白序列與阿拉伯芥(Arabidopsis)的RTE1蛋白有較高的相似性。在先前裕舜學長研究結果發現,ACTG7-1會在病毒感染的早期有表現量下降的現象,而當利用病毒誘導基因靜默(virus induced gene silencing; VIGS)技術降低ACTG7-1在菸草中的表現量時,竹嵌紋病毒的外鞘蛋白的累積量相較於控制組提高了四倍多。為了釐清ACTG7-1是參與了病毒的複製或是移動,利用經VIGS處理後的原生質體(protoplast)進行接種實驗,發現當降低ACTG7-1在細胞內的表現量後,竹嵌紋病毒的外鞘蛋白的累積量相較於控制組提高了2-3倍,顯示ACTG7-1影響在病毒的複製。進而利用real-time定量RT-PCR來量測正負股病毒基因體在細胞內的累積量,推測降低ACTG7-1的表現,主要是增加了負股基因體的複製效率。前人研究中,RTE1在植物荷爾蒙乙烯的訊號傳遞中扮演重要的角色,主要是藉由調控乙烯受體ETR1使乙烯訊號無法傳遞。前人研究也顯示RTE1在阿拉伯芥的表現位置在內質網膜上,因此我構築了ACTG7-1-orange的fusion protein在菸草上表現,並以共軛焦顯微鏡顯示ACTG7-1在確實位於內質網膜上。然而為了進一步確認乙烯是否對BaMV有影響,我利用乙烯前驅物1-aminocyclopropane-1-carboxylic acid (ACC)處理接種病毒後的原生質體,發現乙烯會有抑制BaMV複製的現象,但不是非常顯著。所以我進一步分析ACC處理接種BaMV後的菸葉,發現外鞘蛋白的累積量相較於控制組減少了約30%,顯示乙烯的確會有抑制BaMV感染的情形。因此,ACTG7-1是如何影響BaMV的複製則有待進一步研究。

Bamboo mosaic virus (BaMV) is a single-stranded positive sense RNA virus. It belongs to Potexvirus of Flexiviridae. Since the virus encodes a limit number of genes can not complete its infection cycle by itself, it requires host factors at different stages of the infection, such as translation, replication, and movement. Previously, our lab used cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique to identify the differentially expressed gene fragments from BaMV infected Nicotiana benthamiana plants. One of the downregulated cDNA fragments, ACTG7-1, is an ortholog to REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) of Arabidopsis. According to the previous study, the expression of ACTG7-1 was knocked down by virus-induced gene silencing technique; the coat protein accumulation level of BaMV was increased about 4 folds to that of control plant at 5 days of post inoculation. To examine whether ACTG7-1 is involved in replication step of the BaMV infection cycle, I prepared the ACTG7-1 knockdown protoplasts derived from knockdown plants and inoculated with BaMV viral RNA. Results indicates that the coat protein accumulation is increased to 2-3 folds that of the control protoplasts suggesting a negative role of ACTG7-1 in BaMV replication. Further analysis of the accumulation of BaMV plus- and minus-strand genomic RNAs by real-time RT-PCR suggests the effect on minus-strand RNA synthesis. Transiently expressed the fusion protein of ACTG7-1 with orange in N. benthamiana shows the ER localization by confocal microscopy. To test whether ACTG7-1 affected the replication of BaMV through the ethylene pathway, we have used the ethylene precursor ACC to enhance the ethylene response. The result indicated ethylene might enhance the resistance against BaMV. The relationship of BaMV replication and ACTG-7-1 in N. benthamiana is still needed to be clarified.
其他識別: U0005-1808201215511300
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