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標題: 自台灣洋桔梗分離的蠶豆萎凋病毒-2之特性及其全基因組之選殖及分析
Characterization, complete genome sequence and genetic organization of Broad bean wilt virus-2 isolated from lisianthus in Taiwan
作者: 沈炳男
Shen, Bing-Nan
關鍵字: lisianthus
Broad bean wilt virus-2
出版社: 植物病理學系所
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摘要: 洋桔梗 [Eustoma grandiflorum (Ref.) Shinners] 是一種在全世界廣受歡迎的觀賞花卉植物。1996年,彰化地區栽種的洋桔梗葉片顯現出黃綠色環斑及壞疽病斑病徵,經過分離後得到一病毒分離株,命名為LV-5。在先前的報告中指出由於LV-5可與市售的BBWV-2抗血清反應,因此認為LV-5可能是屬於蠶豆萎凋病毒-2(Broad bean wilt virus-2, BBWV-2)的其中一個分離株。本次研究的目標在於以分子生物學方式提供資料,以確定LV-5病毒分離株為BBWV-2的成員之一,並且將此病毒的全基因組加以選殖定序、分析其生物特性、以及生產針對此病毒的多株抗體。將病毒自感染LV-5病毒分離株之菸草中純化,在電子顯微鏡底下可以觀察到大量直徑約28 nm的球型病毒顆粒。將兔子以純化的病毒進行免疫後,即可獲得多株抗體,並且以ELISA及Western blotting 的方式確認其偵測此病毒之能力。而寄主範圍反應測定結果顯示BBWV-2 LV-5分離株可以感染4個科共17種植物。LV-5的鞘蛋白全長序列經由反轉錄-聚合
Lisianthus [Eustoma grandiflorum (Ref.) Shinners] is a popular herbaceous ornamental plant worldwide. In 1996, a virus isolate LV-5 was obtained from lisianthus plants showing chlorotic and necrotic spots on leaves in Changhua, Taiwan. The virus was previously identified as a potential isolate of Broad bean wilt virus-2 (BBWV-2) since it positively reacted with a commercial polyclonal antibody against BBWV-2. The objectives of this study were to provide the molecular evidence to confirm that the virus isolate LV-5 is indeed a BBWV-2, to clone and sequence the genome of this virus, to characterize the biological properties and generate polyclonal antibody against this virus. Virus particles were purified from infected Nicotiana benthamiana plants and 28 nm isometric virions were observed by an electron microscopy. Polyclonal antibody was produced by immunizing the rabbit with purified virus, and the ability to detect BBWV-2 isolate LV-5 was demonstrated by western blotting and ELISA. The host range reactions test showed that 17 plant species belonging to 4 families were susceptible to BBWV-2 isolate LV-5. Coat protein (CP) genes of LV-5 were amplified by RT-PCR and then cloned and sequenced. Comparisons of the nucleotide sequence of large-CP (L-CP) and small-CP (S-CP) showed that LV-5 shared 77.6-92.3% and 77.1-93.9% identities, respectively, to those of other eighteen BBWV-2 isolates. Comparisons of the amino acid sequence of L-CP and S-CP showed that LV-5 shared 84.8-97.5% and 88.8-98.0% identities, respectively, to those of other BBWV-2 isolates. The L-CP of LV-5 has 61.5-62.3% nucleotide and 62.7-64.4% amino acid identies with those of BBWV-1 isolates. The S-CP of LV-5 has 60.5-62.4% nucleotide and 58.4-59.9% amino acid identities with those of BBWV-1 isolates. Near full-length genomic sequences was obtained via genome walking techniques and the 5´ terminal sequences were obtained from RT-PCR with oligo-d(T) as primers using polyadenylated dsRNA as template. Excluding the 3´ terminal poly-A tails, the RNA 1 and 2 are 5947 nt and 3555 nt, respectively. All genes on both RNAs were determined according to the proposed protease cleavage sites. Comparison of the full-length sequences showed that the LV-5 isolate shared 78.6-93.0% identity on RNA 1 and 78.7-92.0% on RNA 2 to those of other seven published BBWV-2 isolates. Phylogenetic analyses based on the full-length nucleotide sequences of RNA 1 or RNA 2 and the amino acid sequences of two coat proteins showed that LV-5 isolated from lisianthus in Taiwan was closely related to the BBWV-2 IP isolate infecting pepper in Japan. Taken together, our results provide the molecular evidence to confirm that virus isolate LV-5 from lisianthus in Taiwan is an isolate of BBWV-2.
其他識別: U0005-2108200714554200
Appears in Collections:植物病理學系



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