香蕉萎縮病毒輕微病徵型系統之基因體研究

Abstract

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.