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題名 | 感染百子蓮之Nerine virus X病毒之血清學與分子生物學鑑定及其檢測技術之建立=Serological and Molecular Identification of Nerine virus X Infecting African Lily (Agapanthus africanus L.) and Development of Its Detection Techniques |
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作 者 | 江芬蘭; 張清安; 鄭櫻慧; 陳金枝; | 書刊名 | 臺灣農業研究 |
卷期 | 60:2 2011.06[民100.06] |
頁次 | 頁89-100 |
分類號 | 433.4 |
關鍵詞 | 百子蓮; 病毒病; Nerine virus X病毒; 多元抗體; 全長基因體; RT-PCR檢測; African lily; Agapanthus africanus; Virus disease; Nerine virus X; Polyclonal antiserum; Full-length genome; RT-PCR; |
語文 | 中文(Chinese) |
中文摘要 | 本研究於梅峰地區採集獲得葉片呈現黃綠斑駁病徵之百子蓮植株,取其葉片接種於奎藜(Chenopodium quinoa Willd.),經連續3次單斑接種後獲得一分離株(AL-1)。將AL-1回接於百子蓮實生苗植株亦可造成與田間病株類似之斑駁病徵。以接種AL-1之奎藜葉片為材料,利用高低速交替及硫酸銫等密度平衡離心進行病毒純化,純化試料經電子顯微鏡觀察,病毒顆粒為絲狀,長度預估約508 nm。純化病毒經SDS-PAGE電泳分析顯示其鞘蛋白乃由單一種蛋白質基本單位(protein subunit)所構成,分子量估算為25 kDa。將純化之AL-1病毒顆粒注射於白兔後所製備之多元抗體於ELISA (Enzyme-linked immunosorbent assay)、SDS-免疫擴散反應(SDS-immunodiffusion test)及西方轉漬法反應中,可與同源之純化病毒抗原及百子蓮罹病株發生反應。以萃取自接種AL-1之奎藜病葉之全量核酸為模版,利用數組本研究所設計之引子對分別進行病毒全長度基因體之RT-PCR核酸增幅與選殖,定序結果確認AL-1分離株之全長基因體約由6577個核苷酸所組成(不包含polyA),內含5個轉譯架構(ORF1-5)分別對應5種基因產物。其中ORF 2-3含有彼此重疊之序列,此等基因體結構與已知之Potexvirus屬病毒相同。經比對GenBank上已登錄之其他10種potexviruses全長基因體序列後顯示,AL-1基因體之核苷酸與胺基酸序列均與Nerine virus X (NVX)最為相近,相同度高達93%以上。針對AL-1之ORF1及鞘蛋白基因所分別設計之二種引子對,均可以專一性地檢測出AL-1。基於前述AL-1分離株之生物特性、血清學以及全長度基因體之分析結果,證實本研究於呈現黃綠條紋及斑駁病徵之百子蓮所分離之病毒應為NVX病毒之一個系統,此乃NVX於台灣發生之首度報導。 |
英文摘要 | African lily (Agapanthus africanus L.), native in South-Africa, is currently a popular bulbous floral crop in Taiwan and many other countries. An African lily exhibiting foliar chlorotic strip and mottle symptom was collected from Mai-Fung nursery of Taiwan University in 2005. A virus isolate, designated as AL-1, was obtained from this plant by establishing a single lesion isolate in Chenopodium quinoa Willd. Back inoculation of AL-1 onto seedlings of African lilies confirmed that AL-1 was able to induce chlorotic mottle symptom which was similar to what was observed in the field. Virions of AL-1 were successfully purified from the inoculated tissue of C. quinoa by differential and equilibrium centrifugation in cesium sulfate. Elongated virus particles estimated at 508 nm in length were observed from the purified sample under an electron microscope. By SDS-PAGE, molecular weight of AL-1's coat protein was determined as 25 kDa. An antiserum against purified AL-1 virions was prepared in rabbit and shown to react strongly and specifically with its homologous antigens prepared from diseased samples of field-collected or inoculated ones in enzyme-linked immunosorbent assay (ELISA), SDS-immunodiffusion, and western blotting tests. Full-length genome of AL-1 was revealed by RT-PCR amplification with random primer and several primer pairs designed in this study according to those closely related virus sequences in the GenBank. Genome of AL-1 was found to compose 6577 nucleotides, excluding the poly (A) tails, in which five open reading frames (ORF) encoding presumably replicase (ORF 1), triple gene blocks (ORF 2-4) and coat protein (ORF 5) were located. This genome structure was equivalent to that of Potexvirus and most resembled to Nerine virus X (NVX) (Acc. No. AB219105) in the GenBank. The percent identities of nucleotide and amino acid sequences of the full-length genomes between AL-1 and NVX were 98 and 93%, respectively, indicating they were isolates of the same virus. A primer pair for specific detection of AL-1 was designed and shown to be useful in routine RT-PCR test. Our results revealed that AL-1 was an isolate of NVX based on the biological and serological characterizations and the molecular analysis of the full-length genomic sequences. To our knowledgement, it's the first report of NVX occurrence in Taiwan. |
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