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研究生:林冠妤
研究生(外文):Kuan-YuLin
論文名稱:衛星核酸干擾竹嵌紋病毒複製之探討與應用
論文名稱(外文):Investigation and application of satellite RNA-mediated interference with Bamboo mosaic virus replication
指導教授:林納生
指導教授(外文):Na-Sheng Lin
學位類別:博士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
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:2012
畢業學年度:100
語文別:英文
論文頁數:154
中文關鍵詞:竹嵌紋病毒衛星核酸干擾病毒複製複製核酸靜默
外文關鍵詞:Bamboo mosaic virussatellite RNA-mediate interferencereplicationRNA silencing
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台灣超過九成以上叢生型竹類皆被竹嵌紋病毒 (bamboo mosaic virus,
BaMV) 感染以致於竹筍品質下降及產量減少造成經濟上嚴重的損失。BaMV是一種單股正極核酸病毒,而竹嵌紋病毒衛星核酸 (satellite RNA of BaMV,satBaMV)小分子單股正極核酸需BaMV 為協助病毒協助其複製及包被。某些從自然界找到的satBaMV 分離株具有干擾BaMV 複製的特性,例如BSL6。干擾型satBaMV 抑制病毒複製的決定因子已被證實位於其5’端非轉譯區域裡特殊二級結構apical hairpin stem loop 內的一個核苷酸。然而干擾型satBaMV如何抑制病毒複製的機制仍不清楚。近十年來,越來越多證據顯示核酸靜默在病毒複製扮演重要的角色。故本文主要探討核酸靜默對BaMV 及satBaMV 複製之影響及其對干擾型satBaMV 抑制病毒複製之影響。我們使用核酸靜默缺失的植株或過量表達基因靜默中重要酵素因子的植株來進行BaMV 及satBaMV之接種試驗。從結果得知,BaMV 及satBaMV 的累積量在DICER-like (DCL) 2/4及2/3/4 阿拉伯芥突變株中會增加然而在DCL1 突變株中卻會減少。而在RNA-dependent RNA polymerase 6 (RDR6) 靜默的轉殖菸草植株中,在新葉可觀察到BaMV 引發之病徵且BaMV 及satBaMV 的累積量也增加。然而儘管在RDR6 靜默的轉殖株中病毒累積量增加,從BaMV 產生的小片段核酸 (viral small RNAs, vsRNAs) 累積量卻會減少。但從satBaMV 產生的小片段核酸(satBaMV small RNAs, sat-sRNAs) 卻與其累積量呈正相關。此外,儘管之前的文獻都指出在核酸靜默抗病毒的過程中ARGONAUTE 1 (AGO1) 與AGO2 扮演重要的角色,但過量表現AGO2、AGO5 及AGO9 為限制BaMV 複製的重要因子。進一步分析vsRNAs 及sat-sRNAs 得知,大部分的小片段核酸都是由BaMV及satBaMV的正股及反股產生的21 及22 個核苷酸片段,而產生vsRNAs的熱門區域會由於病毒感染的宿主不同而造成差異,但並不受共同感染satBaMV 影響。由於在BaMV 及BSL6 共同接種的植株中僅能偵測微量的vsRNAs,故推論BSL6 在寄主引發核酸靜默之前就已對病毒的複製造成影響。另外,我們建立了表現干擾型satBaMV 的轉殖菸草和阿拉伯芥並證明其對BaMV 具有抗性,而且病毒抗性與轉基因干擾型satBaMV 的表現量呈正相關。故在本論文中,我們確定DCL 2/4 及RDR6 是影響BaMV 及satBaMV 複製的重要因子。從本研究及先前實驗結果(Chen et al, 2012) 可以推論BSL6 是藉由與BaMV 競爭複製酶聚合物而造成對BaMV 複製的影響,由於sat-sRNAs 不會被RDR6 複製而減輕第二波的核酸靜默作用,所以satBaMV 在競爭複製酶聚合物上更具優勢。此外,表現干擾型satBaMV 之轉殖株對BaMV 具有抗性,證明未來防治BaMV 將多了一項新的選項。
Bamboo mosaic virus (BaMV), a single-stranded, positive-sense RNA virus, infects more than 90 % of cultivated bamboos having pachymorph rhizomes in Taiwan bamboo plantations and caused great economic loss. Satellite RNAs of BaMV (satBaMVs), a single-stranded, positive-sense RNA molecules, depend on BaMV for replication and encapsidation. Some satBaMV isolates, such as BSL6, collected from the natural field, interfere with BaMV replication whereas BSF4 does not. The determinants of BaMV interference have been mapped to a single nucleotide in the apical hairpin stem-loop structure of the satBaMV 5’ untranslated region. However, the underlying mechanism is still unclear. Because RNA silencing functions in antiviral defense, the role of gene silencing in BaMV and satBaMV replication or in BSL6-mediated BaMV interference remains invedtigated. Mutants with deleted or overexpressed genes involved in RNA silencing, such as dicer-like (DCL), argonaute (AGO) and RNA-dependent RNA polymerase (RDR) were used to test the effects on BaMV and satBaMV replication and BSL6-mediated BaMV interference in Arabidopsis thaliana and Nicotiana benthamiana. BaMV and satBaMV levels were increased in dcl 2/4 and dcl 2/3/4 but reduced in dcl 1 A. thaliana. In rdr6 N. benthamiana, the accumulation and movement of BaMV and satBaMV were enhanced. Despite the increased level of BaMV in rdr6 N. benthamiana, the level of BaMV small RNAs (vsRNAs) were lower than that in BaMV-infected wild-type plants. However, the level of satBaMV small RNAs (sat-sRNAs) were related to the satBaMV level. It was known that AGO1 and AGO2 play important roles in antiviral defense, and we found overexpression of AGO 2, 5 and 9 could downregulate BaMV replication. BSL6 mediated the repression of BaMV replication in all the DCLs, AGO1 and RDR6 knockdown mutants. Global analyses of BaMV and satBaMV small RNA profiles revealed that most vsRNAs and sat-sRNAs are 21 and 22 nt in length from both positive and negative polarities. The vsRNAs hot-spot regions differ in N. benthamiana and A. thaliana but not with satBaMV co-inoculation. Only few amount of vsRNAs were detected from plants co-infected with BaMV and BSL6 demonstrated that BSL6 interferes with BaMV replication may take place before RNA silencing. Moreover, BaMV-resistant transgenic N. benthamiana and A. thaliana plants expressing interfering satBaMV were successfully generated, which confers more resistance to viral RNA than with virion infection. The resistance of BSL6 transgenic plants was positively associated with the transcript level of the transgene. Together with previous results (Chen et al, 2012), our studies suggest that BSL6 interferes in BaMV replication through competition for replication complexes with BaMV. In summary, DCL 2/4 and RDR6 are involved in restricting BaMV and satBaMV replication, and satBaMV achieves preferential replication by avoiding RDR-dependent silencing. I propose a model of BSL6-mediated interference of BaMV through replication complex competition. The interfering satBaMV transgenic plants show BaMV resistance, which could be applied in bamboo biotechnology for control of BaMV.
Abstract I
中文摘要 III
Acknowledgements V
List VI
List of Tables VIII
List of Figures X
Contents 1
1 Introduction 1
1.1 Subviral RNAs associated with plant viruses 1
1.2 Effects of satRNAs on helper viruses 1
1.3 Bamboo mosaic virus (BaMV) 3
1.4 Satellite RNA of BaMV (satBaMV) 4
1.5 Host factors involved in BaMV and satBaMV replication 5
2 Objectives 7
3 Chapter 1: Role of gene silencing in BaMV and satBaMV replication and BSL6-mediated BaMV interference 8
3.1 Introduction 8
3.2 Material and methods 11
3.3 Results 16
3.4 Discussion 19
4 Chapter 2: Global analysis of BaMV and satBaMV small RNAs in different hosts 23
4.1 Introduction 23
4.2 Material and methods 24
4.3 Results 26
4.4 Discussion 35
5 Chapter 3: Expression of interfering satellite RNA in transgenic plants confers resistance to BaMV 45
5.1 Introduction 45
5.2 Material and methods 47
5.3 Results 49
5.4 Discussion 54
6 Conclusion and perspectives 59
Reference 61
Appendix 129
1 Interactions of host, BaMV and satBaMV 129
1.1 Introduction 129
1.2 Material and methods 129
1.3 Results and discussion 131
2. Curriculum Vitae 154

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