請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/36178
標題: 雙生病毒於原核及真核系統複製機制之研究
Studies on the Replication Mechanisms of Geminiviruses in Prokaryotic and Eukaryotic Systems
作者: 吳佳穎
Wu, Chia-Ying
關鍵字: Geminivirus
雙生病毒
Replication Mechanism
Infectious Clone
Prokaryotes
複製機制
感染性選殖株
原核生物
出版社: 生物科技學研究所
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摘要: 已知雙生病毒在包括植物界、酵母菌以及原核生物具有跨物種複製之能力。為了研究雙生病毒在真核以及原核細胞中的複製機制,建立容易操作的病毒複製系統是必需的。在病毒的分子生物學方面的基礎研究與病毒載體等應用性研究皆為必要且深具挑戰性的工作。欲了解病毒的基因功能、基本生活史、與致病機制等資訊,必須先構築具有感染力的選殖株,才能進行突變、重組等基礎研究。先前的研究中多需採用(部分)多套體的構築方式,以多重步驟的限制酵素截切、黏合、與再選殖,方能完成構築。本研究為簡化此流程以提高雙生病毒感染性選殖株之構築效率,採用滾環式擴增法(Rolling Circle Amplification, RCA),配合限制酵素不完全切割,直接產生雙生病毒基因多套體之選殖株。經由接種測試,證實藉由此方法所獲得的三種雙生病毒雙套體選殖株,分別為霍香薊黃脈病毒南投分離株 (Ageratum yellow vein virus Nan-Tou isolate, AYVV-NT 又稱為 AYVTV-NT),番茄捲葉病毒台南分離株 (Tomato leaf curl virus Tainan isolate, TLCV-TN) 以及南瓜捲葉病毒雲林分離株 (Squash leaf curl virus Yunlin isolate, SqLCV-YL),皆具有62.5~100 %的感染效率。此方法大幅縮減產生雙生病毒感染性選殖株所需的時間與多次黏合、選殖所耗費的人力與物力。預期此法將對於其他具有環狀基因體病毒的基礎與應用性研究具有極大助益。 多套基因體病毒通常需要在同一細胞中聚積所有的致病因子才能起始感染。對於雙生病毒而言,雙基因體病毒在感染效率上並不具有特別優勢。然而在演化過程中,單基因體之雙生病毒並不是絕對多數。為了探討單基因體與雙基因體雙生病毒在感染效率上的優劣關係。沿用上述構築雙生病毒感染株的方法,可將雙基因體之南瓜捲葉病毒 (SqLCV-YL) 改造成單基因體之嵌合體(DNA A+B chimera),所得到的單基因體感染性選殖株在此稱為 pBinSqLAB10。為了證明單基因體選殖株較雙基因體選殖株具有較好的感染效率。運用十倍系列稀釋的方法以等量的農桿菌接種源進行植物感染效率測試。當接種源十倍稀釋時,兩者之感染效率皆為 100% ;但是當稀釋因子提高為一千倍時,則雙基因體感染選殖株之感染效率下降為 0%。 以上結果顯示單基因體感染性選殖株確實增強了雙基因體雙生病毒的感染效率。此外,在所有接種單基因體選殖株的植物中,經由反向聚合?反應(inverse PCR) 可測得一環狀缺失性的 DNA (defective DNA),經過連續追蹤發現此缺失性 DNA 可穩定累積在感病植物中。將所有反向聚合?反應產物經由割膠純化、選殖以及解序得知此環狀缺失性 DNA 之組成有一致性,且為部份序列的DNA A+B 嵌合體,其環化的位置在單一共有的複製起始點。推測此缺失性 DNA 的產生是單基因體病毒選殖株在植物中進行滾環式複製(rolling circle replication; RCR) 時所衍生出的。由以上結果提供了直接的分子證據,說明了單位長度的病毒基因體從多套體病毒選殖株上被釋放出來是以滾環式複製的機制,而不是以同源性重組的方式。本研究首次展示了藉由人為方式構築所得到的單基因體選殖株確實增強了雙基因體雙生病毒的感染力,未來將有助於多套基因體病毒之高效率病毒載體之研發。另一方面,藉由環狀缺失性嵌合體的產生提供直接的證據說明雙生病毒在真核系統中的複製機制。 此外,為了研究雙生病毒在原核物種中的複製機制。 本研究以霍香薊黃脈病毒屏東株系 (Ageratum yellow vein virus Ping-Dong isolate, AYVV-PD) 為材料,利用大腸桿菌噬菌體 M13 之基因體特性以及兩者複製機制的相似性,將一個單位長度的雙生病毒基因體構築於噬菌體基因體中來建立雙生病毒之原核型複製系統。在此系統中,以南方墨點分析法測得雙生病毒基因體專一性之訊號,且分別與病毒基因體之病毒股和互補股探針反應,但是不與噬菌體探針反應。經由更精密之二維洋菜膠體電泳分析(2D-AGE)以及限制酵素處理後此訊號仍然存在,證明此訊號為單股基因體意即雙生病毒之病毒股以及互補股基因體同時存在。為了進一步證實為環狀基因體,設計專一性之引子對進行反向聚合?反應 (inverse PCR),結果如預期之片段長度且接點正確。此外, 將另外兩種雙生病毒:石薯嵌紋病毒 (Gonostegia mosaic virus) 以及番茄捲葉病毒 (TLCV) 的基因體也構築於此系統,都可測得病毒基因體的產生。而以相似基因體長度的胡瓜嵌紋病毒 2a 基因當作負對照組未測得2a 基因體的產生。上述結果說明了噬菌體 M13 的複製系統確實具有支援雙生病毒環狀基因體產生之能力並可利用此原核系統來研究雙生病毒之複製機制。進一步探討雙生病毒的基因是否參與產生病毒單基因體的過程,將雙生病毒複製相關蛋白之基因 (Rep gene) 進行突變則無法測得單股環狀基因體的產生。利用二維蛋白質電泳和 Rep 蛋白專一性血清,可測得此雙生病毒在原核複製系統中確實有 Rep 蛋白之表現,以上結果證明在此原核系統中必須具有功能性的 Rep 蛋白才足以支援產生雙生病毒單股環狀基因體。 研究雙生病毒如何在已分化完全的植物細胞中起始複製是個重要的議題。雙生病毒不具有複製酵素且已分化完全的細胞中其細胞核內不再進行 DNA 複製。根據前人研究以及雙生病毒可於原核系統中進行複製的相關報導,我們假設原核型胞器-葉綠體可能為病毒起始複製的地點。本研究以 AYVV 為例,設計並利用特定股專一性聚合?反應(strand-specific PCR) 來偵測病毒複製型態 DNA。由實驗結果得知此方法具有專一性且可在純化的感病植物葉綠體中測得極微量的病毒複製型態基因體。另一方面,為了觀察病毒複製相關蛋白是否存在於葉綠體中,構築具有感染力並且可在植物細胞中表現綠色螢光蛋白的病毒基因體重組株。在接種植物後,利用螢光顯微鏡初步觀察,可於少數葉肉細胞中觀察到綠色螢光的訊號。未來可利用雷射掃描共軛焦顯微鏡觀察植物活體細胞中葉綠體是否有螢光訊號來證明病毒複製相關蛋白可能存在於葉綠體中。將可提供證據說明葉綠體的原核型複製系統是否可以支援雙生病毒基因體的合成。 整體研究的結果有助於了解雙生病毒在真核及原核細胞中的複製機制也暗示了不同的 DNA 複製機制可能具有共同的演化來源以及雙生病毒在演化過程中保留了利用不同 DNA 複製機制的能力。另一方面,發展簡易方法構築雙生病毒多套體感染選殖株的研究,將可應用於選殖其他具有多套環狀基因體的病毒,並且有助於高效率病毒載體之研發。
Infectious clones of viruses are essential tools in both the basic and applied researches in virology. Most infectious clones of geminiviruses consist of (partial) tandem repeats of viral genomes in the vectors, which usually involve tedious, multi-step assemblies of genomic fragments during the constructions. To enhance the efficiency, a simplified procedure was devised in this study, which employed limited restriction digestions of the multimeric viral genomes produced by rolling circle amplification (RCA), followed by direct cloning into the proper vectors. The efficiency of the procedure and the infectivity of the dimeric constructs were demonstrated using three different geminiviruses, Ageratum yellow vein virus Nan-Tou isolate (AYVV-NT equal to AYVTV-NT), Tomato leaf curl virus Tainan isolate (TLCV-TN), and Squash leaf curl virus Yunlin isolate (SqLCV-YL). This procedure simplifies significantly the construction of multimeric infectious clones of geminiviruses, and may further be applied in both basic and practical research to viruses with circular genomes. Viruses with segmented genomes suffer from the constraint that all segments must enter the same infection foci to initiate a successful infection. However, there is no apparent preference for monopartite genomes in the evolutionary history of begomoviruses. To compare the infection efficiency among geminiviruses with monopartite or bipartite entities, construction of a monopartite infectious clone of bipartite Squash leaf curl virus (SqLCV) was conducted. By limited digestion of rolling circle amplification (RCA) products, the tandem DNA A and B repeats of SqLCV was generated, pre-ligated, and further cloned into pBin19 vector to give the monopartite chimera named as pBinSqLAB10 Agroinfection of equivalent inoculums harboring mono- or bi-partite infectious clones with the decimal series dilutions (101~103), the infection efficiency of bipartite construct dropped drastically, but the high infection rates maintained on pBinSqLAB10-inoculated plants. Moreover, small defective DNAs derived from pBinSqLAB10-inoculated plants were detected by inverse PCR and accumulated stably dependent on the machineries of host plants. Through the cloning and sequencing analyses, all inverse PCR products reveled the constancy on consisting of partial chimeric sequence of DNA A and B compassing with a cognate replication origin. These data illustrated that the unit-length genomes of geminiviruses released from tandem-repeated construct were through the RCR mechanism rather than homologous recombination. This report firstly demonstrated that the construction of artificial monopartite chimera enhanced the infectivity of bipartite geminiviruses and presented the feasibility on creation of efficient viral vectors for multipartite viruses with segmented genomes. Replication of genomic DNAs of plant-pathogenic begomoviruses has been demonstrated in prokaryotes, which supported the possibility of analyzing DNA replication process of begomoviruses in bacteria. However, previous studies indicated that the replication of begomovirus DNAs in prokaryotes requires tandem constructs of viral genomes with at least two copies of the origin of replication (ori). Since the rolling circle replication (RCR) systems of geminiviruses and phage M13 share substantial similarities, it is possible that the machinery of phage M13 may assist the replication of geminiviruses in bacteria. In this study, phage M13 vector harboring the unit-length genome with only a single copy of ori of a monopartite begomovirus, Ping-Dong strain of Ageratum yellow vein virus (AYVV-PD), was constructed and used to investigate the replication of AYVV-PD DNAs in Escherichia coli JM101. The generation of single-stranded, circular DNAs (sscDNAs) corresponding to the unit-length AYVV-PD genome of both polarity was observed and verified by Southern blot analyses, nuclease digestions and two-dimensional neutral/alkaline agarose gel electrophoreses (2D-AGE). Replication-associated protein (Rep) of AYVV-PD was detected only in bacteria generating the corresponding sscDNAs, whereas disruption of the Rep gene abolished the phenomenon. The results suggested that a single copy of ori is sufficient for the prokaryotes to support the generation of unit-length, genomic sscDNAs of begomoviruses, which requires the presence of functional Rep protein. Initiation of replication in differentiated host cells that have already exited S-phase of cell cycle has been an important subject of researches for geminiviruses. As known the prokaryotic-type machineries supported the replication of begomoviruses, we proposed that self-proliferating, prokaryotic-type organelle, chloroplasts, might be the proper location for initiating the geminiviruses replication when viruses invaded the non-dividing cells. In this study, the replicative forms of viral DNA could be detected by strand-specific PCR (Sts-PCR) in purified chloroplasts of AYVV-infected plants. This implies that geminiviruses possible to do replicate in the bacterial-descended chloroplasts. Alternatively, the infectious recombinants (Rep-eGFP or Cp-eGFP) expressed the viral proteins fused with enhanced green fluorescence protein were constructed. After co-agroinfected the Nicotiana benthamiana, small portion of mesophyll cells of the symptomatic leaf (epinasty) expressed the eGFP proteins visualized by fluorescence microscope. In the future, this system will apply to observe the viral proteins subcellular localizations in vivo by using confocal. The observations of the viral proteins associated with the replication of geminivirus DNAs in chloroplasts may provide additional information on geminivirus life cycles and evolution. Taken together, these studies suggested a common origin of evolution among different types of DNA replication machineries and indicated that the geminiviruses might have maintained the ability to utilize diverse DNA replication systems including eukaryotic- and prokaryotic-type machineries during the evolutionary process.
URI: http://hdl.handle.net/11455/36178
其他識別: U0005-2207200811063100
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2207200811063100
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