Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30971
標題: 蝴蝶蘭上新potyvirus全長度基因體序列之選殖及分析
Complete genome sequence and genetic organization of a new potyvirus infecting Phalaenopsis orchids
作者: 翁梨娟
Weng, Li-Chuan
關鍵字: 蝴蝶蘭
Phalaenopsis
馬鈴薯Y群病毒
Potyvirus
出版社: 植物病理學系所
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Isolation, identification and characterization of a tospovirus causing chlorotic necrosis and ringspot on moth orchids (Phalaenopsis spp.). Joint Annual Meeting of the American Phytopathological Society, Canadian Phytopathological Society, and Mycological Society of America, Québec City, Québec, Canada, July 29– August 2, 2006. Phytopathology 96: Sxx Zheng, Y.-X., Weng, L.-C., Chen, C.-C., and Jan, F.-J. 2005. Molecular evidence that the virus causing chlorotic spots on Phalaenopsis orchids is a new potyvirus. Plant Pathol. Bull. 14: 296-297. Zheng, Y.-X., Yeh, S.-D., Chen, C.-C., and Jan, F.-J. 2003. Molecular cloning of a tospovirus infecting Phalaenopsis orchids in Taiwan. Plant Pathol. Bull. 12: 203-204.
摘要: 於2003年十月,在台灣中部地區所栽培的蝴蝶蘭植株上發現疑似病毒感染所引起的病徵,於葉片上產生黃化斑點病徵,經電子顯微鏡觀察及3′端基因體解序,顯示此一引起蝴蝶蘭黃斑病徵之病毒為一新發現之馬鈴薯Y群病毒(potyvirus),並暫時命名為蝴蝶蘭黃化斑點病毒(Phalaenopsis chlorotic spot virus, PhCSV)。本研究的目的即為將此病毒全長度基因體選殖、解序、分析其基因體組成及生物學特性,並製備可以偵測此一病毒之單株抗體。由被PhCSV感染的圓葉菸草(Nicotiana benthamiana)所純化的病毒顆粒,在電子顯微鏡下觀察呈現直徑約11-12 nm、長約750-800 nm的長絲狀病毒顆粒,經蛋白質電泳(sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE)分離後,可見一大小約33 kDa的鞘蛋白。以純化的病毒對實驗小鼠進行腹腔注射,經免疫反應後摘取小鼠的脾臟進行單株抗體(monoclonal antibody)的生產,同時收取其血液製備多元抗體(polyclonal antibody),針對PhCSV以此老鼠多元抗體測試後具專一性的反應,而在融合瘤細胞株(hybridoma cell)部份亦已篩選到針對PhCSV具靈敏且專一性反應的13個細胞株,這些細胞株將以系列稀釋(limited dilution)方式加以單株化,期能獲得反應良好之單株抗體。在生物特性上為了分析PhCSV的寄主範圍,在本研究中以機械接種的方式測試了來自八個不同科的二十七種不同植物,發現在紅藜(Chenopodium amaranticolor)、綠藜(C. murale)、奎藜(C. quinoa)的接種葉上可觀察到有黃色單斑,而在千日紅(Gomphrena globosa)、奎藜、圓葉菸草及番茄(Lycopersicoum esculentum)上則有系統性病徵的產生。除在生物及血清學上分析外亦於病毒分子層次上進行探討,即針對PhCSV全長度基因體序列進行選殖。本研究中比對多種馬鈴薯Y群病毒序列後設計了三條簡併式引子(degenerated primers),針對PhCSV除了3′端及5′端部分以外的基因體序列,以反轉錄聚合酶鏈鎖反應(reverse transcriptase-polymerase chain reaction, RT-PCR)進行增幅及選殖,而基因體5′端部分則藉由分離所得並經多聚腺苷酸反應(polyadenylated)之雙股RNA為模板,利用已知序列設計所得引子搭配oligo-d (T)進行RT-PCR增幅選殖,經解序獲得之PhCSV基因體不包含3′端poly (A) 的全長度為9804個核苷酸,序列分析發現其上有一個長9237個核苷酸的轉譯架構(open reading frame, ORF),可轉譯出一個長3079個胺基酸的大蛋白(polyprotein);基因體組成與其他馬鈴薯Y群病毒屬病毒相似,核苷酸相同度(nucleotide identity)約為51.6-62.0%、各蛋白胺基酸相同度(amino acid identity)約為20.0-74.0%,相同度最高者均為甜菜嵌紋病毒(Beet mosaic virus, BtMV);經演化樹分析亦指出,在其他馬鈴薯Y群病毒屬病毒中與PhCSV演化關係最為相近者即為甜菜嵌紋病毒。本研究是第一個針對感染蝴蝶蘭的馬鈴薯Y群病毒屬病毒之基因體進行解序及組成分析的研究。
In October 2003, virus-like symptoms of chlorotic spots were observed on leaves of Phalaenopsis orchids in the central part of Taiwan. Results of electron microscopy and the cloning and sequencing of the 3'-genome indicated that the virus isolated from Phalaenopsis orchid is a potyvirus-like virus. The Phalaenopsis potyvirus is tentatively designated as Phalaenopsis chlorotic spot virus (PhCSV). The objectives of this study are to clone, sequence and elucidate the genome organization of PhCSV, to characterize the biological properties and generate antibodies against this virus. PhCSV virions were purified from virus-infected Nicotiana benthamiana plants and found to be long flexuous filaments measuring about 11-12×750-800 nm under an electron microscope. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified virus preparations revealed a structural polypeptide of about 33 kDa. Monoclonal antibody (MAb) against PhCSV was proposed to be generated by immunizing mouse with purified virus particles. After four times of intraperitoneal injection, 400 μl of mouse polyclonal antibodies reacted positively to PhCSV-infected leaf extracts was collected. The spleen cells harvested from the mouse were fused to myeloma cells. Thirteen hybridoma cell lines revealed positive reactions to PhCSV were obtained and are currently being further screened by limiting dilution. In addition, 27 plant species from 8 families were mechanically inoculated for host range assay. Systemic symptoms were observed on Gomphrena globosa, Chenopodium quinoa, N. benthamiana, and Lycopersicum esculentum plants, and chlorotic local lesions were found on inoculated leaves of C.amaranticolor, C. murale, and C. quinoa. In order to clone and obtain the entire genomic sequences of PhCSV, three degenerate forward primers for potyviruses were designed. Three distinct cDNA fragments covering most of the PhCSV genome except the 5'- and 3'- termini were obtained by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate forward primers combined with the primer extension. The sequences of 5'-termini were obtained from RT-PCR using oligo-d (T) as primer with the polyadenylated dsRNA as template. The complete genome of PhCSV comprises 9804 nucleotides in length, excluding the 3' terminal poly (A) tail. Computer analysis of the sequence revealed a large open reading frame (ORF) that commences at the first AUG at nucleotide position 189 and terminates at nucleotide 9425, encoding a single polyprotein of 3079 amino acid residues excluding 3'-noncoding region of 379 nucleotides. The genome organization of PhCSV was found to be similar to those of potyviruses sharing a complete genome nucleotide identity ranging from 51.6% to 62.0%, with the highest identity (62.0%) to Beet mosaic virus (BtMV). Comparison of amino acids of each protein on PhCSV genome with those of other potyviruses shared 20.0% to 74.0% amino acid identity, having highest identity (74.0%) with BtMV. The phylogenetic analysis of the polyprotein of PhCSV with those of the other viruses in the Potyvirus revealed that the PhCSV is closely related to BtMV. This is the first report of a complete genome sequence of the potyvirus infecting Phalaenopsis orchids.
URI: http://hdl.handle.net/11455/30971
其他識別: U0005-2808200608592500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2808200608592500
Appears in Collections:植物病理學系

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