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題 名 | 宮燈百合Gloriosa stripe mosaic virus之核酸分子和免疫法鑑定=Molecular and Serological Identification of Gloriosa stripe mosaic virus on Christmas Bells (Sandersonia aurantiaca Hook) |
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作 者 | 陳金枝; 江芬蘭; 黃春惠; | 書刊名 | 臺灣農業研究 |
卷 期 | 67:3 2018.09[民107.09] |
頁 次 | 頁229-246 |
分類號 | 435.456 |
關鍵詞 | 宮燈百合; 嘉蘭條斑嵌紋病毒; 全長度核酸序列; RT-PCR檢測; 多元抗體; Christmas bells; Sandersonia aurantiaca Hook; Gloriosa stripe mosaic virus; GSMV; Full-length sequence; RT-PCR detection; Polyclonal antibody; |
語 文 | 中文(Chinese) |
中文摘要 | 本研究於進口宮燈百合(Sandersonia aurantiaca Hook)檢測出一種Potyvirus屬病毒,經鞘蛋白(coatprotein; CP)核酸序列分析,確認其乃屬於嘉蘭條斑嵌紋病毒(Gloriosa stripe mosaic virus; GSMV)成員,分離株代號CB。CB分離株以反轉錄-聚合酶鏈鎖反應(reverse transcription-polymerase chain reaction; RT-PCR)法由其核酸3'端之polyA起始,往5'端以不同引子對分段增幅,進而選殖其核酸片段進行定序,共解得9,455個核苷酸,其內包含對應全長度大蛋白之9,156個核苷酸(對應3,052個胺基酸),相關序列已登錄於GenBank並取得登錄序號為EF427894。對應CB分離株之CP有798個核苷酸,可轉譯出266個胺基酸。本研究由宮燈百合和火焰百合(即嘉蘭) (Gloriosa spp.)另分別定序得到5個GSMV分離株的CP核苷酸序列,並均登錄於GenBank取得序號,包括來自宮燈百合的CB4 (KT764122)和CB18 (KT764123);來自火焰百合的GL2 (KT764120)、GL3 (KT764124)和GL4 (KT764121)。所有分離株與國外已登錄之火焰百合GSMV分離株(EU042761)間,CP胺基酸序列相同度介於86.5-94.0%,均屬於GSMV成員。而由類緣關係顯示,來自3個宮燈百合GSMV分離株間有較相近的分群。依據CB-CP核苷酸序列設計3組引子對(上游引子分別為GSMV715u、GSMV660u及GSMV500u,搭配共同下游引子GSMVd),以GSMV660u/GSMVd及GSMV500u/GSMVd兩組引子對,於RT-PCR法中均可檢測出宮燈百合和火焰百合上之GSMV;而GSMV715u/GSMVd僅可檢測到宮燈百合GSMV,對感染火焰百合之GSMV則無預期片段被增幅出,此引子對可應用於宮燈百合GSMV之鑑別性檢測。將CB-CP全長度核苷酸選殖於表現載體pET28b上,轉型於Escherichia coli strain Rosetta (DE3)宿主內誘導表現蛋白之生成,將預估分子量約31.6 kDa之細菌表現蛋白純化後,經兔免疫注射而獲得對應CB-CP之多元抗體。於indirect enzyme-linked immunosorbent assay (ELISA)和西方墨點法中,此CB多元抗體可與同源抗原(CB-CP表現蛋白)產生反應,也可成功檢出宮燈百合和火焰百合之GSMV罹病組織。GSMV為文獻上發生於火焰百合之病毒記錄,但於宮燈百合上為首次發現,本研究並為首度完成GSMV全長度核酸定序者。本研究所製備之GSMV引子對及多元抗體,均已應用於進口宮燈百合種球之病毒監測,可提升我國對GSMV之自主檢測能力。 |
英文摘要 | A Potyvirus virus (isolate code CB) was detected from an imported bulb of Christmas bells (Sandersonia aurantiaca Hook), and further identified as the Gloriosa stripe mosaic virus (GSMV) based on the sequences of the coat protein (CP) coding region. The full-length genome sequence of CB was determined, revealing that the CB genome is 9,455 nucleotides (nt) long (accession number EF427894), encoding a polyprotein with 3,052 amino acids (aa). The fragment of 798 nt is coding for 266 aa of the CB CP. Moreover, the CP nt sequences of another five virus isolates, namely CB4 (KT764122) and CB18 (KT764123) from Christmas bells, and GL2 (KT764120), GL3 (KT764124), and GL4 (KT764121) from gloriosa, were also determined and shared 86.5-94.0% aa identities with that of the known GSMV-CP (EU042761) in GenBank, indicating that they are also GSMV isolates. The phylogenetic analysis indicated that the CB, CB4, and CB18 from Christmas bells are closely related. Three primers sets (upstream primers: GSMV7150u, GSMV660u and GSMV500u, and a common downstream primer: GSMVd) were designed from the CB-CP sequence for detection of GSMV. The primer pairs GSMV660u/GSMVd and GSMV500u/GSMVd were useful to amplify the expected amplicons from all of GSMV isolates from Christmas bells and gloriosa in reverse transcription-polymerase chain reaction (RT-PCR). The primer pair GSMV715u/GSMVd can amplify the expected 715- bp fragment only from the GSMV isolates of Christmas bells, and is useful for distinguishing those GSMV isolates of Christmas bells from gloriosa’s. The full-length nucleotides of the CB-CP were constructed to the expression vector pET28b, and transformed into Escherichia coli strain Rosetta (DE3) for induction of the overexpressed viral protein. Polyclonal antiserum against the CB-CP, prepared by immunizing the bacterial expressed fusion protein into a rabbit, positively reacted with the GSMV-CP in the virus-infected Christmas bells and gloriosa tissues in indirect enzyme-linked immunosorbent assay (ELISA) and western blotting. In this report, GSMV was first found on Christmas bells, and its full-length genome sequence was first completed. The prepared GSMV primer pairs and polyclonal antiserum are helpful to strengthen our ability in GSMV inspection. |
本系統中英文摘要資訊取自各篇刊載內容。