臺灣博碩士論文加值系統

香蕉萎縮病毒 (Banana bunchy top virus, BBTV) 及香蕉條紋病毒 (Banana streak Mysore virus, BSMysV) 為感染香蕉的兩種DNA病毒，分別造成香蕉萎縮病以及香蕉條紋病。BSMysV一般以游離型存在；但也嵌入了拔蕉 (Musa balbisiana) 基因體中，使得許多栽培種的香蕉帶有不具感染性的嵌入型BSMysV。而嵌入型病毒序列可能因逆境或壓力而活化成具有感染性的病毒，因此BSMysV除了藉游離型感染香蕉外，嵌入型也有活化而造成病害的風險，因而影響了香蕉生產。植物體能夠將胞嘧啶進行甲基化以調控序列活化狀態。過去發現一些DNA病毒於植物體中發生甲基化，有些病毒甲基化的比例和寄主植物病徵嚴重程度有關。故本研究利用亞硫酸氫鹽定序分析BBTV及BSMysV甲基化狀態，藉此了解這兩種香蕉DNA病毒是否誘發基因靜默。BBTV基因體的DNA-R及DNA-M甲基化比例均低，顯示BBTV未引發寄主產生強烈的基因靜默。比較病徵嚴重型及輕微型之病毒DNA甲基化平均比例後發現病毒甲基化之比例無法反映其病徵嚴重程度。而由於心葉及老葉中的病毒DNA甲基化比例無明顯差異，顯示不同病程發展時期中的病毒甲基化的情形無明顯差異。游離型BSMysV僅發生輕微甲基化，顯示未誘發強烈的基因靜默；但嵌入型BSMysV明顯發生了甲基化，CG及CHG類型甲基化之比例顯著高於CHH類型，推測嵌入型BSMysV可能發生基因靜默而處於不活化的狀態。但於嵌入型BSMysV中偵測到ORF III部份區域甲基化比例極低，推測嵌入型BSMysV仍有可能活化而造成病害。

Banana bunchy top virus (BBTV) and Banana streak Mysore virus (BSMysV) are DNA viruses that causing Banana bunchy top disease and Banana streak virus disease in bananas (Musa spp.), respectively. Generally, BSMysV presents as a free virus, an episome, in hosts. However, BSMysV has integrated into the genome of M. balbisiana, thus, many banana cultivars contain non-infectious BSMysV (eBSMysV). Endogenous pararetrovirus sequences may be activated to become infectious due to stress. BSMysV have an impact on the banana production because of not only its episomal form infecting bananas directly but also eBSMysV which is likely to become infectious. In plants, cytosine methylation is one of the ways to regulate the level of activation of DNA. Some DNA viruses were shown to be methylated by host plants, and among these viruses, the levels of methylation in several viruses are related to the symptom severity. In this study, to determine whether DNA viruses in the banana were transcriptionally silenced, we analyzed the methylation patterns of BBTV and BSMysV by bisulfite sequencing. The methylation levels of DNA-R and DNA-M of BBTV were quite low, and it indicated that BBTV did not induce strong transcriptional gene silencing. By comparing the average percentages of methylation between severe strain and mild strain of BBTV, it showed that the methylation levels of BBTV did not reflect the symptom severity. The difference in the level of methylation between the heart leaf and the old leaf was not statistically significant, so it suggested that the methylation levels of BBTV did not change drastically in different pathogenesis stages. For BSMysV, episomal BSMysV is hypomethylated and doesn’t induce strong transcriptional gene silencing. On the contrary, eBSMysV is hypermethylated, and the methylation rate is significantly higher in symmetric (CG and CHG) than in asymmetric (CHH) cytosines. It indicated that eBSMysV may be transcriptionally silenced. However, there was almost no methylation observed in a partial region of ORF III in eBSMysV, so we speculated that eBSMysV is probable to be activated.

誌謝 ii
摘要 iii
Abstract iv
目錄 vi
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1 香蕉 1
1.2 香蕉病毒病害 2
1.2.1 重要病毒病害簡介 2
1.2.2 香蕉DNA病毒病害 4
1.3 寄主植物利用基因靜默 (gene silencing) 以防禦病毒侵染 8
1.4 病毒抵抗基因靜默的方式 9
1.4.1 RNA病毒 9
1.4.2 DNA病毒 10
1.5 本研究之目的 11
第二章 材料與方法 12
2.1 植株來源 12
2.2 病毒核酸甲基化之分析 12
2.2.1 香蕉植株核酸萃取 12
2.2.2 菸草核酸萃取 13
2.2.3 製備無甲基化質體pDNA R 13
2.2.4 限制酶酵解反應 14
2.2.5 亞硫酸氫鹽 (bisulfite) 處理 14
2.2.6 設計病毒序列甲基化分析之引子對 15
2.2.7 聚合酶鏈鎖反應（PCR） 15
2.2.8 病毒序列選殖（TA cloning） 16
2.2.9 序列分析 19
第三章 結果 21
3.1 亞硫酸氫鹽處理效力測定 21
3.2 BBTV甲基化分析 22
3.2.1 BBTV DNA-R甲基化分析 22
3.2.2 BBTV DNA-M甲基化分析 22
3.3 BSMysV甲基化分析 23
第四章 討論 25
第五章 參考文獻 31
第六章 圖表 42

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