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研究生:黃薇頻
研究生(外文):Wei-Pin Huang
論文名稱:於菸草原生質體中追踪竹嵌紋病毒核酸基因體的研究
論文名稱(外文):Tracing Bamboo Mosaic Virus RNA Molecules in Nicotiana benthamiana Protoplasts
指導教授:蔡慶修蔡慶修引用關係
指導教授(外文):Ching-Hsiu Tsai
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:2008
畢業學年度:96
語文別:英文
論文頁數:43
中文關鍵詞:竹嵌紋病毒核酸基因體細胞分佈噬菌體鞘蛋白噬菌體RNA序列
外文關鍵詞:Bamboo mosaic virusRNAsub-cellular localizationMS2 coat proteinMS2 RNA sequence
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竹嵌紋病毒(Bamboo mosaic virus)為單一正股RNA病毒,隸屬於Flexiviridae科之Potexvirus屬,具有五個主要的轉譯區,分別可以轉譯出病毒生命週期所必須的蛋白。雖然已經有許多研究protexvirus複製的文獻發表,但是關於病毒RNA於細胞座落位置與複製間關係的研究卻很缺乏。即使有許多可以透過光學顯微鏡操作來觀察細胞內RNA位置的方法,但它們大多只侷限於觀察固定後的樣品。近年來已建立一個可以在活體樣本中追踪標定RNA的實驗方法,其策略是利用噬菌體MS2的外鞘蛋白會與其 RNA具有專一性結合之能力來進行實驗。因此,本實驗的目的就是利用此方法來觀察竹嵌紋病毒在細胞內的相關位置。首先,必需將噬菌體MS2 RNA序列插入BaMV 之核酸基因體中,以提供與MS2蛋白結合的位置;另外構築MS2鞘蛋白、螢光蛋白(GFP)及核定位訊號(NLS)之融合蛋白,當此融合蛋白和修飾後的BaMV共同接種後,藉由偵測螢光蛋白的位置來追蹤標定BaMV之相對位置,而未和目標核酸結合之自由GFP-NLS -CPMS2融合蛋白則將進入細胞核以減少偵測時之背景值。首先我們將修飾後之竹嵌紋病毒接種於菸草原生質體來測試其複製效率,結果發現,不管插入幾個重覆的MS2 RNA序列於竹嵌紋病毒的3''端非轉譯區之前皆會影響竹嵌紋病毒基因體之累積,但並不會影響次基因體之累積。另一方面,將GFP-NLS -CPMS2融合蛋白構築於pBI 121質體中,並進行功能性測試。GFP-NLS -CPMS2融合蛋白與HcPro混合利用農桿菌進行短時間地表現後以共軛焦螢光顯微鏡觀察,而由實驗結果得知此GFP-NLS -CPMS2融合蛋白可在菸草葉下表皮細胞裡正常表現,並且所有的GFP-NLS -CPMS2融合蛋白會座落於細胞核中與預期結果相符合。之後將修飾好的BaMV.S.(MS2)6接種於GFP-NLS -CPMS2融合蛋白表達之菸草原生質體,接種一天後進行GFP-NLS -CPMS2融合蛋白位置的觀測,而GFP-NLS -CPMS2融合蛋白的位置即表示竹嵌紋病毒基因體於細胞內座落的位置。觀測結果顯示GFP的螢光訊號可以與被染劑標定之粒線體座落一起,並且可以隨著動態的粒線體一起移動。經由這個實驗方法我們可以觀測到竹嵌紋病毒基因體座落於細胞內的位置,雖然由先前的複製測試結果顯示目前我們觀測到GFP訊號大部份是代表竹嵌紋病毒次基因體於細胞內座落的位置,而非竹嵌紋病毒基因體於細胞內座落的位置。
Bamboo mosaic virus (BaMV), a potexvirus, is a single-strand, positive-sense RNA virus which contains five open reading frames (ORFs) which encode proteins for replication, movement and structure and a caped 5''UTR and a 3''UTR with a poly A tail. There is good amount of information on the replication of potexviruses but information on the sub-cellular localization of the viral RNA and the site of replication is lacking. Though there are different methods to detect RNA localization by optical microscopy, most of them are limited to fixed samples. We employ a technique which exploits the ability of phage MS2 coat protein to bind specifically to only its cognate RNA. In this strategy, the MS2 RNA sequence is inserted into target RNA (BaMV genomic RNA) and MS2 coat protein is fused with green fluorescent protein (GFP) which contains a nuclear localization signal (NLS). When the modified target RNA and the GFP-NLS-CPMS2 construct are co-transformed into protoplasts the GFP-NLS-CPMS2 fusion protein binds to the target RNA and the green fluorescence signal indicates the localization of the target RNA. The unbound GFP-NLS-CPMS2 is retained in the nucleus due to the NLS thereby reducing noise. This method can be used for four dimensional study of RNA localization in live cells. First, the MS2 RNA sequence had to be inserted into BaMV RNA genome. According previous study, the PVX 3''UTR insertion construct and only subgnomic RNAs could accumulate normally. Simultaneously, the CPMS2-GFP fusion protein was constructed in pBI 121 vector. As BaMV.S.(MS2)6 was transfected in GFP-NLS-CPMS2 fusion transiently expressed protoplasts1 dpi, observation of GFP localization indicated the BaMV RNA subcellular localization. GFP signal was co-localized and moved with mobile mitochondria stained with Mitotracker OrangeCMTMRos. Employing this novel method we observed BaMV RNA localizing at the mitochondria and that a large proportion of the RNA would most probably be the subgenomic RNAs which also contains the MS2-hps since the mutant RNAs are defective in genomic RNA accumulation.
中文摘要......i
Abstract......iii


Introduction

Bamboo mosaic virus......1
Replication of BaMV......2
A novel technique established for the direct real-time visualization of RNA localization......3


Materials and Methods

Construct of pBaMV.S.(MS2)n......6
Construct of GFP-NLS-CPMS2 fusion protein.....7
Agrobacterium cultures and plant infiltration......8
In vitro transcription......9
Protoplast preparation, inoculation, and transfection......9
Northern blotting and western blotting......10
4'',6-diamidino-2-phenylindole (DAPI) staining......10
Mitotracker Orange CMTMRos staining......11
GFP visualization by confocal microscopy......11


Result

Description of the components and the course of this study.......12
Insertion of six repeats of CPMS2 RNA sequence preceding the 3’UTR of BaMV.S affects BaMV genomic RNA replication but not sub-genomic RNA synthesis.......13
Visualization GFP signal confirms the functionality of GFP-NLS-CPMS2 fusion protein.......14
GFP-NLS-CPMS2 fusion protein was localized in nucleus of pBI/GNC transfected N. benthamiana protoplasts in the absence of MS2-hps.......15
Replication of BaMV.S.(MS2)6 in protoplast can relocate GFP-NLS-CPMS2 from the nucleus to mitochondria.......15

Discussion......17

Table and Figures
Table 1......25
Table 2......25
Fig.1......26
Fig.2......27
Fig.3......28
Fig.4......29
Fig.5......30
Fig.6......32
Fig.7......33
Fig.8......34
Fig.9......35

Rferences......36
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