Genome characterization of the mild strain of Banana bunchy top babuvirus

香蕉萎縮病是由香蕉萎縮病毒(Banana bunchy top babuvirus, BBTV)所引 起，受害香蕉會產生植株萎縮、葉脈透化、葉片萎黃細小並叢集於株頂、中肋與 葉柄處顯現濃綠色條斑，甚至不結果等病徵。BBTV 為多基因體之單股環狀DNA 病毒，目前已發現其基因體至少包含六條約1.1 kb 的DNA components，分別為 DNA 1 ~ 6，其中DNA 1 為負責病毒複製的master replication initiation protein (master Rep)；而在某些香蕉病株上尚可分離出能夠自行複製約1.1 kb 的additional Rep-encoding components。根據香蕉寄主上病徵型之不同可將BBTV 區分為五種 系統(type I ~ V)，而此五種系統亦可利用三對保守性引子對－C1, S 與SR 進行 PCR 檢測之結果加以鑑定區分。其中type V 之病徵型為輕微型系統(mild strain)， 目前僅在台灣被報導，但其基因體組成尚未被完整研究，因此本論文針對此分離 株之基因體組成，利用保守性與專一性引子對，對其總核酸進行PCR 反應，增 幅出可能存在之基因體並建構其PCR library，再以restriction fragment length polymorphism (RFLP)分析之結果進一步對選殖株進行分群，並將不同RFLP 群進 行定序及序列分析。由結果顯示自type V 分離株V-1 與V-2 中均可得到DNA 1 ~ 5，卻無法找到DNA 6。然而以南方墨點分析無法由此二分離株之總核酸中偵測 到DNA 3 與DNA 6 專一性探針之訊號，顯示輕微病徵型統分離株之BBTV 基因 體含量可能甚低，故無法以南方墨點分析偵測。以即時定量聚合酶連鎖反應 (real-time PCR)偵測DNA 1 與DNA 3 之結果顯示，輕微病徵型系統分離株之DNA 1 與DNA 3 含量較嚴重型病徵型系統分離株低1000 倍以上，因此推測type V 可 能缺少DNA 6、其序列與已知之DNA 6 相似度太低或是其含量甚少，無法以現 有之方法偵測到。相較於其他BBTV 系統，type V 具有約0.5 Kb 的S 引子對之 PCR 產物，經由選殖與定序分析得知其在V-1 與V-2 二分離株中均為defective master Rep，並可依其序列與基因體結構再區分為五群，由免疫捕捉PCR (Immunocapture PCR)之結果推測這些defective master Rep 能夠被病毒鞘蛋白包 被；此外在V-2 分離株中則可另外發現一約0.7 Kb 之defective master Rep 存在。 然而DNA 6 之不存在或是defective Rep 之存有是否可能影響BBTV type V 之病 徵抑制，尚待進一步的研究確定。The causal agent of Banana bunchy top disease is Banana bunchy top babuvirus (BBTV). The BBTV-infected banana plants show symptoms of dwarf, bunchy top, leaf atrophy, vein clearing, dark-green streak on pseudostem, and were unable to produce fruits in severe cases. BBTV is a complex circular single-stranded DNA virus consist of six genomic integral components, DNA 1 ~ 6. DNA 1 encodes master replication initiation protein (master Rep) for viral genomic replication. Besides master Rep, components encode additional replication initiation protein (additional Rep) with the self-only-replication function could also be isolated from some BBTV-infected banana plants. BBTV strains can be classified into five types, type I ~ V, defined by symptoms, and could be differentiated by polymerase chain reaction (PCR)-based assay using C1, S and SR primer pairs. Type V is a mild strain, and its complete genome organization has yet been characterized. In this study, primer pairs based on conserved and specific regions of BBTV components were designed. The designed primer pairs and total nucleic acids extracted from each type V isolate was used in PCR reaction. The PCR products were cloned to construct PCR-libraries. Clones from each PCR-library were analyzed by restriction fragment length polymorphisms (RFLP). Randomly selected clones from each RFLP group were sequenced. Based on result of analysis, five integral components, DNA 1 ~ 5, were identified from isolates of type V, V-1 and V-2. However we were unable to obtain DNA 6 from both isolates even two primer pairs designed from DNA 6 conserved region. No signal could be detected from total nucleic acids extracted of V-1 or V-2 isolates by Southern blot analysis using DNA 6 open reading frame as probes. However we were also unable to detect DNAs in the nucleic acids by Southern blot analysis. It indicates that the amounts of BBTV genomic DNA are much lower in the mild strains. Real-time quantitative PCR were performed to quantify the amounts of DNA 1 and DNA 3. The result indicated the amount of DNA 1 and DNA 3 in mild strain isolates were fewer than in severe strain isolates by more than 1000 folds. These data suggest that DNA 6 was absent or had some sequence changes in mild strain. Besides, mild strain had a ca. 0.5 kb PCR product amplified by S primer pairs. After cloning and sequencing of these fragments, we found that they are defective forms of master Reps, and they could be classified into five subgroups by sequence homologies and genome organizations. The result of immunocapture PCR suggests these defective Reps are incapsidated. In addition to the 0.5 kb defective master Reps, another 0.7 kb defective master Rep could also be identified from V-2 isolate. Further studies are needed to resolve if the absence of ordinary DNA 6 and/or the presence of defective Rep could account for symptoms amelioration of mild strain virus infection.中文摘要........................................................................................................................1 英文摘要........................................................................................................................3 壹、緒論........................................................................................................................5 貳、前人研究..................................................................................................................8 一、香蕉萎縮病之發生歷史與地理分布................................................................8 二、香蕉萎縮病之病原探討....................................................................................9 三、香蕉萎縮病之病徵紀錄.................................................................................10 四、香蕉萎縮病之傳播途徑與發病生態..............................................................11 五、香蕉萎縮病毒基因體之研究..........................................................................12 六、香蕉萎縮病毒病徵系統之分群......................................................................22 參、材料與方法............................................................................................................25 一、健康與罹病香蕉之來源..................................................................................25 二、輕微與嚴重分離株之病徵紀錄......................................................................25 三、病毒分離株之分子特性鑑定..........................................................................25 (一)酵素連結免疫吸附分析(Enzyme-linked immunosorbent assay, ELISA)... .................................................................................................................26 (二)香蕉總核酸之抽取...................................................................................27 (三)聚合酶連鎖反應 (Polymerase chain reaction, PCR)...............................27 (四)洋菜膠體電泳分析(Electrophoresis analysis)......................................... 27 四、輕微病徵系統之基因體選殖、分群與定序..................................................28 (一)聚合酶連鎖反應.......................................................................................29 (二)連合反應(Ligation).................................................................................. 29 (三)大腸桿菌Top10F’與XL1-Blue勝任細胞之製備.....................................29 (四)大腸桿菌Top10F’與XL1-Blue 勝任細胞之轉形....................................30 (五)菌落聚合酶連鎖反應(Colony PCR)........................................................30 (六)限制片段長度多型性分析(Restriction fragment length polymorphism, RFLP).........................................................................................................31 (七)質體少量製備(Mini-prep)........................................................................31 (八)DNA定序..................................................................................................32 (九)DNA與所預測胺基酸序列之分析..........................................................32 五、以南方墨點分析(Southern blot analysis)偵測BBTV基因體.........................33 (一)探針之製備...............................................................................................33 (二)核酸之雜合與轉漬...................................................................................34 (三)雜合訊息之偵測.......................................................................................35 六、以即時絕對定量聚合酶連鎖反應偵測各病徵型分離株基因體含量..........36 七、以IC-PCR 檢測BBTV基因體是否為鞘蛋白所包被....................................37 肆、結果........................................................................................................................39 一、BBTV 嚴重與輕微病徵型系統之病徵差異比較...........................................40 二、各BBTV 病徵型系統之分子特徵差異比較..................................................40 (一)酵素連結免疫吸附分析(ELISA).............................................................40 (二)聚合酶連鎖反應(PCR).............................................................................40 三、BBTV 輕微病徵型系統基因體之選殖...........................................................41 (一) Integral components 之選殖.....................................................................41 1. 以C1 引子對進行integral component 之選殖................................41 2. 以S 引子對進行integral component 之選殖...................................42 3. 以DNA 2 專一性引子對進行DNA 2 之選殖.................................42 4. 以DNA 6 專一性引子對進行DNA 6 之選殖.................................42 5. 基因體與序列分析所用integral component 之選擇......................43 (二) Defective Rep 之選殖...............................................................................43 1. 0.5 kb defective Rep 之選殖............................................................43 2. 基因體與序列分析所用0.5 kb defective Rep 之選擇....................43 3. 0.7 kb defective Rep 之選殖............................................................44 4. 基因體與序列分析所用0.7 kb defective Rep 之選擇....................44 (三) Additional Rep 之選殖與分群.................................................................44 1. 以SR引子對進行additional Rep 之選殖........................................45 2. 以S1 引子對進行additional Rep 之選殖......................................45 3. 以S2 引子對進行additional Rep 之選殖........................................46 4. 以S3 引子對進行additional Rep 之選殖........................................46 5. 以Y引子對進行additional Rep 之選殖..........................................46 6. 基因體與序列分析所用additional Rep 之選擇.............................47 四、BBTV 輕微病徵型系統基因體之分析...........................................................47 (一) Integral component 部分...........................................................................47 1. 台灣各病徵型分離株間之親緣演化分析......................................47 2. DNA 1 之序列與親緣演化分析.....................................................48 3. DNA 2 之序列與親緣演化分析......................................................49 4. DNA 3 之序列與親緣演化分析......................................................49 5. DNA 4 之序列與親緣演化分析......................................................50 6. DNA 5 之序列與親緣演化分析......................................................51 7. V-1 與V-2 分離株之CR-SL序列分析與二級結構比較................52 8. V-1 與V-2 分離株之CR-M序列分析..............................................52 (二) Defective Rep 部分...................................................................................53 1. 0.5 kb Defective Rep 之序列、基因體結構與親源演化分析.........53 2. 0.7 kb Defective Rep 之序列與基因體結構分析...........................55 3. Defective Rep 之CR-SL 序列分析與二級結構預測......................56 4. Defective Rep 之CR-M序列分析...................................................57 (三) Additional Rep-encoding component 部分...............................................57 1. Additional Rep 之核苷酸序列分析與核苷酸親源演化分析.........57 2. Additional Rep 之胺基酸序列分析與胺基酸親源演化分析.........58 五、以南方墨點分析偵測BBTV 基因體..............................................................58 六、以即時定量聚合酶連鎖反應偵測BBTV 基因體含量..................................59 (一) DNA 1 之絕對定量..................................................................................60 (二) DNA 3 之絕對定量..................................................................................60 (三)各病徵型系統分離株之DNA 1 與DNA 3 含量比較..............................61 七、以免疫捕捉聚合酶連鎖反應偵測鞘蛋白所包被之BBTV 基因體..............61 伍、討論........................................................................................................................63 陸、圖表........................................................................................................................74 表一、聚合酶連鎖反應所用引子之相關資訊......................................................74 表二、序列分析所用BBTV integral components 之相關資訊.............................76 表三、進行序列分析所用BBTV additional components 之相關資訊.................78 表四、BBTV分離株type II-8、V-1 與V-2 於北蕉之病徵表現.............................79 表五、使用單元抗體2H6 以直接酵素連結免疫吸附分析法測定各BBTV 分離 株中病毒顆粒含量....................................................................................80 表六、BBTV V-1 與V-2 分離株之integral components 組成，核苷酸與預測之 胺基酸長度................................................................................................81 表七、各BBTV分離株間DNA 1 核苷酸序列全長成對比較..............................82 表八、各BBTV分離株間DNA 1 胺基酸序列全長成對比較..............................84 表九、各BBTV分離株間DNA 2 核苷酸序列全長成對比較..............................86 表十、各BBTV分離株間DNA 2 胺基酸序列全長成對比較..............................87 表十一、各BBTV分離株間DNA 3 核苷酸序列全長成對比較..........................88 表十二、各BBTV分離株間DNA 3 胺基酸序列全長成對比較..........................90 表十三、各BBTV分離株間DNA 4 核苷酸序列全長成對比較..........................92 表十四、各BBTV分離株間DNA 4 胺基酸序列全長成對比較..........................93 表十五、各BBTV分離株間DNA 5 核苷酸序列全長成對比較..........................94 表十六、各BBTV分離株間DNA 5 胺基酸序列全長成對比較..........................95 表十七、BBTV V-1、V-2、MTC1 與MTC2 分離株之defective replication initiation proteins (Rep) 組成與核苷酸長度..............................................96 表十八、各BBTV 輕微病徵型系統分離株間defective Rep 核苷酸序列全長成 對比較....................................................................................................97 表十九、BBTV V-1 與V-2 分離株之additional Rep 組成，核苷酸與預測之胺 基酸長度................................................................................................99 表二十、各BBTV分離株間additional Rep 核苷酸序列全長成對比較............100 表二十一、各BBTV分離株間additional Rep 胺基酸序列全長成對比較........103 表二十二、以即時定量聚合酶連鎖反應偵測BBTV V-1 與V-2 分離株DNA 1 之含量...........................................................................................107 表二十二、以即時定量聚合酶連鎖反應偵測BBTV V-1 與V-2 分離株DNA 3 之含量...........................................................................................108 圖一、嚴重型與輕微型系統分離株之矮化與束頂病徵差異............................109 圖二、嚴重型與輕微型系統分離株葉脈透化程度之病徵表現差異................110 圖三、嚴重型與輕微型系統分離株假莖出現深綠色條斑之病徵表現差異....111 圖四、以C1、S 與SR 引子對或S1、S2、S3 與Y 專一性引子對，對各 BBTV 分離株總核酸進行聚合酶連鎖反應...........................................112 圖五、BBTV I-1、II-1、IV、V-1 與V-2 分離株之基因體DNA component 全長核苷酸序列親源演化分析..............................................................113 圖六、BBTV I-1、II-1、IV、V-1 與V-2 分離株之基因體DNA component 全長胺基酸序列親源演化分析..............................................................114 圖七、BBTV I-1、II-1、IV、V-1 與V-2 分離株之DNA 1 選殖株核苷酸 序列全長排比..........................................................................................115 圖八、BBTV I-1、II-1、IV、V-1 與V-2 分離株之DNA 1 選殖株胺基酸 序列全長排比..........................................................................................118 圖九、BBTV DNA 1全長核苷酸序列親源演化分析........................................119 圖十、BBTV DNA 1全長胺基酸序列親源演化分析........................................120 圖十一、BBTV V-1 分離株之V-1-1b 與V-1-1Hung 核苷酸與胺基酸序列全長 排比.......................................................................................................121 圖十二、BBTV I-1、II-1、IV、V-1 與V-2 分離株之DNA 2 選殖株核苷酸 序列全長排比.......................................................................................123 圖十三、BBTV I-1、II-1、IV、V-1 與V-2 分離株之DNA 2 選殖株胺基酸 序列全長排比.......................................................................................127 圖十四、BBTV DNA 2 全長核苷酸序列親源演化分析....................................128 圖十五、BBTV DNA 2 全長胺基酸序列親源演化分析....................................129 圖十六、BBTV I-1、II-1、IV、V-1 與V-2 分離株之DNA 3 選殖株核苷 酸序列全長排比.............................................................................130 圖十七、BBTV I-1、II-1、IV、V-1 與V-2 分離株之DNA 3 選殖株胺基 酸序列全長排比.............................................................................133 圖十八、BBTV DNA 3 全長核苷酸序列親源演化分析....................................134 圖十九、BBTV DNA 3 全長胺基酸序列親源演化分析....................................135 圖二十、BBTV I-1、II-1、IV、V-1 與V-2 分離株之DNA 4 選殖株核苷酸 序列全長排比.................................................................................136 圖二十一、BBTV I-1、II-1、IV、V-1 與V-2 分離株之DNA 4 選殖株胺基 酸序列全長排比.............................................................................138 圖二十二、BBTV V-1-4、V-2-4、I-1-4 與II-1-4 之transmembrane domain 之預測圖.........................................................................................139 圖二十三、BBTV DNA 4 全長核苷酸序列親源演化分析................................140 圖二十四、BBTV DNA 4 全長胺基酸序列親源演化分析................................141 圖二十五、BBTV I-1、II-1、IV、V-1 與V-2 分離株之DNA 5 選殖株核苷 酸序列全長排比.............................................................................142 圖二十六、BBTV I-1、II-1、IV、V-1 與V-2 分離株之DNA 5 選殖株胺基 酸序列全長排比.............................................................................144 圖二十七、BBTV DNA 5 全長核苷酸序列親源演化分析................................145 圖二十八、BBTV DNA 5 全長胺基酸序列親源演化分析................................146 圖二十九、BBTV V-1、V-2、MTC1與MTC2分離株基因體內DNA components 於stem-loop common region (CR-SL)之核苷酸序列排比...........147 圖三十、BBTV V-1 分離株CR-SL之二級結構預測..........................................148 圖三十一、BBTV V-2 分離株CR-SL之二級結構預測......................................150 圖三十二、BBTV V-1 分離株基因體之DNA components 於major common region (CR-M)核苷酸序列排比........................................................152 圖三十三、BBTV V-2 分離株基因體之DNA components 於major common region (CR-M)核苷酸序列排比..........................................................153 圖三十四、BBTV V-1、V-2、MTC1 與MTC2 分離株之defective Rep 選殖株 苷酸序列排比.................................................................................154 圖三十五、BBTV 0.5 kb defective Reps 基因體結構.........................................159 圖三十六、BBTV V-1、V-2、MTC1 與MTC2 分離株中defective Rep 選殖株 全長核苷酸序列親源演化分析........................................................161 圖三十七、BBTV V-2 分離株中V-2-7Da 與V-2-7Db 之基因體結構...............162 圖三十八、BBTV MTC1 分離株CR-SL之二級結構預測.................................163 圖三十九、BBTV MTC2 分離株CR-SL之二級結構預測.................................164 圖四十、BBTV MTC1 分離株基因體之DNA components 於major common region (CR-M)核苷酸序列排比..........................................................165 圖四十一、BBTV MTC2 分離株基因體之DNA components 於major common region (CR-M)核苷酸序列排比..........................................................166 圖四十二、BBTV additional Rep 全長核苷酸序列親源演化分析....................167 圖四十三、BBTV additional Rep 全長胺基酸序列親源演化分析....................168 圖四十四、以南方雜合分析偵測BBTV II-8、IV、V-1 與V-2 分離株之總 核酸.................................................................................................169 圖四十五、以DNA 1-Q 與DNA 3-Q 引子對增幅的PCR 產物之核苷酸序列 .........................................................................................................170 圖四十六、Ct 值與BBTV DNA 1 與DNA 3 質體倍率稀釋之線性關係圖 .........................................................................................................171 圖四十七、以DNA 1-Q、DNA3-Q 與Musa Radka4 引子對，對各BBTV 分離 株總核酸進行聚合酶連鎖反應.....................................................172 圖四十八、BBTV 罹病株與健康Cavendish 香蕉樣品之擴增曲線圖與解離曲 線圖.................................................................................................173 圖四十九、BBTV 罹病株與健康Cavendish 香蕉樣品之擴增曲線圖與解離曲 線圖.................................................................................................175 圖五十、以C1、S 與SR 引子對對各BBTV 分離株總核酸進行免疫捕捉聚合 酶連鎖反應........................................................................................177 柒、參考文獻..............................................................................................................178 捌、附錄......................................................................................................................183 附錄一、V-1 分離株之C1 基因體庫....................................................................183 附錄二、V-2 分離株之C1 基因體庫....................................................................188 附錄三、V-1 分離株之S 基因體庫......................................................................191 附錄四、V-2 分離株之S 基因體庫......................................................................198 附錄五、V-1 分離株之D2 基因體庫....................................................................205 附錄六、V-2 分離株之D2 基因體庫....................................................................208 附錄七、V-1 分離株之S5 基因體庫....................................................................213 附錄八、V-2 分離株之S5 基因體庫....................................................................220 附錄九、MTC1 分離株之S5 基因體庫................................................................223 附錄十、MTC2 分離株之S5 基因體庫................................................................227 附錄十一、V-2 分離株之S7 基因體庫................................................................233 附錄十二、V-1 分離株之SR基因體庫................................................................236 附錄十三、V-2 分離株之SR基因體庫................................................................240 附錄十四、V-2 分離株之S1 基因體庫................................................................242 附錄十五、V-2 分離株之S2 基因體庫................................................................244 附錄十六、V-2 分離株之S2 基因體庫................................................................249 附錄十七、V-1 分離株之Y基因體庫..................................................................253 附錄十八、V-2 分離株之Y基因體庫..................................................................25