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標題: 番茄黃化捲葉病毒複製相關蛋白 (Rep) 與病毒基因體 DNA 之交互作用
The interaction between replication-associated protein (Rep) and viral genomic DNA of Tomato yellow leaf curl virus
作者: 黃彥華
Huang, Yan-Hua
關鍵字: 豆類金黃嵌紋病毒屬;Geminivirus;番茄黃化捲葉病毒;複製相關蛋白;滾環式複製作用;基因間區域;DNA 結合區域;拓樸異構酶;begomovirus;tomato yellow leaf curl virus (TYLCV);replication-associated protein (Rep);rolling circle replication (RCR);intergenic region (IR);DNA-binding domain;topoisomerase
出版社: 生物科技學研究所
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番茄黃化捲葉病毒 (Tomato Yellow Leaf Curl Virus, TYLCV) 為雙生病毒科,豆類金黃嵌紋病毒屬的單股環狀DNA病毒,其基因間區域 (intergenic region, IR),包含複製起始點以及雙向的啟動子。複製相關蛋白 (replication-associated protein, Rep) 是雙生病毒在進行DNA複製時所不可缺少的蛋白,此Rep蛋白會辨認IR上專一性的病毒DNA序列 (iterons),並於virion-sense上具有高度保留性的TAATATT↓AC序列進行剪接,藉此啟動雙生病毒的滾環式複製機制 (rolling-circle replication, RCR)。而本篇研究目的則在於探討TYLCV之Rep蛋白與其基因體DNA之交互作用機制,確認iterons序列及位置,並對Rep蛋白與iterons結合之區域及其第一型異構酶活性進行分析。首先利用滾環式擴增法 (Rolling Circle Amplification, RCA) 將TYLCV基因體從病毒的傳播媒介-菸草粉蝨 (Bemisia tabacci) 的蟲體內複製出來,配合限制酵素之截切,再選殖到pUC119載體上,接著利用PCR技術,分別擴增完整的Rep 基因以及Rep的N端 (Rep1-181) 和C端 (Rep177-361) 序列,並且選殖到pET21d和pET29a表現載體上,進行蛋白之大量表現,接著以割膠後電泳回收的方式,純化目標蛋白,並且利用此Rep蛋白製備Rep專一性血清。之後以此血清配合西南方墨點法 (Southwestern blot),分別由DNA與蛋白質方向探討Rep蛋白和病毒DNA間的交互作用機制。在DNA結構方面,結果證實Rep蛋白可專一性辨認雙股環狀的雙生病毒DNA,但並無法與線狀的病毒DNA結合,顯示Rep蛋白辨識目標DNA的方式包含結構之差異。在DNA序列方面,發現Rep蛋白也會在in vitro的情況下,優先與TYLCV之IR、Rep DNA與C4 DNA之間發生結合作用,此情形同樣也發生在Rep1-181蛋白上,此結果也暗示Rep和C4 編碼序列上可能有iterons存在。而在Rep蛋白之剖析方面,本研究發現全長的Rep蛋白與Rep1-181對於病毒DNA具有結合能力,而Rep177-361蛋白則可能為結合之專一性的決定因子。在Rep蛋白活性方面,模擬病毒在in vitro的環境下,進行滾環式複製作用,並且在複製的過程中,偵測Rep蛋白的第一型拓樸異構酶功能,結果顯示Rep蛋白在此條件下確實存在切割DNA的活性 (nicking activity),證實由原核系統表現出的Rep蛋白,仍然保有其原本的活性;此外,配合利用Φ29 DNA polymerase進行滾環式複製反應,亦可觀察到經由Rep蛋白的接合活性 (re-ligation activity) 所產生的單股環狀DNA產物。綜上所述,本研究完成TYLCV Rep蛋白與病毒DNA間交互作用的基礎分析,定位出Rep蛋白與病毒DNA交互作用的區間與結構,並研發經由細菌所大量表現、純化且具有酵素活性Rep蛋白的流程。期能藉由本研究而增進對於雙生病毒DNA複製機制之了解,並發展截斷TYLCV病毒複製循環的方法,以應用於被其他雙生病毒感染的植物上,達到治療雙生病毒疾病的目標。

Tomato Yellow Leaf Curl Virus (TYLCV), a member of the family Geminiviridae, genus Begomovirus, harbors single-stranded circular DNA genome. The intergenic region of TYLCV genomic DNA contains bidirectional promoter elements interspersed within the origin of replication. The replication-associated protein (Rep) is essential for viral DNA replication, in which the Rep protein initiates rolling circle replication by binding to specific repeated sequences (iterons) within the origin of replication and acting as a topoisomerase. The aim of this study is to explore the underlying mechanism for the specific interaction between TYLCV Rep protein and the cognate viral genomic DNA by identifying the sequence and location of TYLCV iterons and DNA-binding domains of TYLCV Rep protein. The TYLCV genome was amplified from Bemisia tabacci (whiteflies) by Rolling Circle Amplification (RCA), and cloned into the pUC119 vector. The full length Rep open reading frame, N-terminus (Rep1-181) and C-terminus (Rep177-361) of Rep were cloned in pET21d and pET29a vectors and expressed in Escherichia coli. The target proteins were purified by electro-elution from polyacrylamide gels, and used to raise Rep-specific antibodies in rabbits. The interactions between Rep proteins and TYLCV genomic DNA were analyzed by Southwestern blot with Rep-specific antibodies. Preliminary results showed that Rep protein specifically interacted with double-strand circular geminivirus DNAs, instead of the linear form DNAs, suggesting that the binding specificity is partially determined by DNA topology. Both full-length Rep and Rep1-181 preferentially interact with IR and the coding regions of Rep and C4 protein, indicating that the main DNA-interacting domain locates at the N-terminus of Rep protein, while Rep177-361 might play the role of specificity determinant. The Type I topoisomerase function of Rep protein were analyzed by a mimicry RCA in vitro by using Φ29 DNA polymerase. The result confirmed that Rep protein possesses nicking / re-ligation activity, providing evidence that the native activity of the Rep protein expressed and purified from prokaryotic cells was still preserved. Through this study, it is expected that practical methods to disrupt the DNA replication cycles could be developed as the effective therapeutics for viral diseases caused by TYLCV and other geminiviruses.
其他識別: U0005-0608201014574700
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