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研究生:余中鼎
研究生(外文):Jong-Ding Yu
論文名稱:探討菸草Argonautes對竹嵌紋病毒感染之影響
論文名稱(外文):The study of Argonautes in Nicotiana benthamiana involved in the infection of Bamboo mosaic virus
指導教授:蔡慶修蔡慶修引用關係
口試委員:林納生徐堯煇鄭綺萍
口試日期:2016-07-05
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:31
中文關鍵詞:菸草竹嵌紋病毒AGO蛋白
外文關鍵詞:Nicotiana benthamianaBamboo mosaic virusArgonautes
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竹嵌紋病毒(Bamboo mosaic virus, BaMV),分類上屬於Alphaflexiviridae科中的Potexvirus屬。是一個單一核酸的正股RNA病毒,全長約有6400個核苷酸。本研究利用本氏菸草(Nicotiana benthamiana)做為病毒寄主,探討植物Argonaute基因與竹嵌紋病毒之間的關係。真核生物具有RNAi(RNA interference)機制對抗入侵的病原體包括病毒,RNAi意指小片段核酸可以影響細胞的基因表現,最典型的方式為加速mRNA的分解或抑制其轉譯,因此植物在病毒入侵細胞後便利用RNAi系統抑制及消除病毒。通常寄主利用RNAi的系統來對抗病毒基因體的機制發生在RNA病毒複製的過程產生了雙股RNA,導致ribonuclease III-like protein (植物體或稱 Dicer-like (DCL))將其切割成21~26核苷酸長的小片段RNA,稱作siRNA(small interfering RNA),這些siRNA再與Argonaute (AGO)及一系列相關蛋白形成RNA-induced silencing complexes (RISCs)去專一性的切割病毒的核酸基因體。在阿拉伯芥(Arabidopsis thaliana)中,已有10個AGO基因被報導,分別為Ago1~Ago10,而在本氏菸草中,目前序列已發表有AGO1, AGO2, AGO4, AGO5等。本研究將利用已知的資訊,設計引子來合成不同的AGO基因片段,並利用病毒引發之基因靜默技術(virus-induced gene silencing, VIGS),使其基因表現量下降,然後觀察這些基因的表現量下降,是否會對竹嵌紋病毒的感染造成影響。實驗結果發現,當AGO4的表現量下降後,竹嵌紋病毒在接種葉及系統葉上的外鞘蛋白的累積皆無明顯的影響,推測AGO4可能沒有參與到植物對抗竹嵌紋病毒的機制中。而在AGO1表現量下降後,竹嵌紋病毒在接種葉及接種原生質體上的外鞘蛋白累積有增加,AGO2表現量下降後則對病毒累積量沒有明顯影響。另外我們也發現在AGO1表現量下降後,AGO2的表現量有上升,因此未來的研究方向將針對AGO1以及AGO2在抵抗竹嵌紋病毒的方式上以及AGO1及AGO2之間的關係進行更進一步的研究。

Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to the genus Potexvirus of the family Alphaflexiviridae. The genome of BaMV is about 6.4 kb with a 5?cap and a 3?poly (A) tail. In this study we use Nicotiana benthamiana as a model plant to inspect the relationship between Argonautes (AGOs) and BaMV. RNA interference (RNAi) in Eukaryotes is known to play an antiviral defense role especially the RNA viruses. During the replication of viral RNAs, the double-stranded (ds) RNA accumulates and triggers the host silencing process. DICER-LIKE (DCL) proteins cleave the viral dsRNA into short interfering RNAs (siRNAs) of 21~24 nucleotides in length. These siRNAs are loaded into AGOs to form the RNA induced silencing complex (RISC). The RISC contains the specific siRNAs and targets to the viral RNAs. There are 10 AGOs identified in Arabidopsis thaliana. By contrast, only four AGOs (AGO1, AGO2, AGO4, and AGO5) were sequenced in N. benthamiana. In this thesis, I used Tobacco rattle virus (TRV)-based gene silencing technique to knock down the expression of different AGOs in N. benthamiana to reveal the relationship between AGOs and BaMV. The results indicated that reducing the expression of AGO4 did not have any effect on the accumulation of BaMV. The results also imply that AGO4 may not play a key role against BaMV in N. benthamiana. By contrast, the reduction of the expression of AGO1 could result in an increase of the accumulation of BaMV in plants and protoplasts. Furthermore, but the accumulation of BaMV has no significant change in AGO2-silencing plants. Moreover, the expression of AGO2 is increased in AGO1-knockdown plants. The results suggest that AGO2 might be controlled by AGO1 as that revealed in Arabidopsis. The relationship between AGO1 and AGO2 in N. benthamiana needs to be further explored.

中文摘要 i
Abstract ii
Introduction 1
Bamboo mosaic virus 1
RNA interference 2
Argonautes 3
Aim 4
Materials and Methods 5
Argonautes knockdown and virus infection. 5
Viral particle purification 6
Viral RNA extraction 6
Protoplast isolation and viral RNA inoculation 7
Western blotting assay 8
Total RNA extraction 8
Real-time RT-PCR 9
Results 11
AGO4 of N. benthamiana do not play any role in BaMV accumulation 11
AGO1 of N. benthamiana has a defense role on BaMV infection 11
AGO2 of N. benthamiana has no significant role in regulating the infection of BaMV 12
The expression of AGO2 in N. benthamiana is regulated by AGO1 13
Discussion 14
Figures 16
References 26


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