宮燈百合Gloriosa stripe mosaic virus 之核酸分子和免疫法鑑定

陳金枝; 江芬蘭; 黃春惠; Chin-Chih Chen; Fen-Lang Chiang; Chun-Huey Huang; 陳金枝;江芬蘭;黃春惠; Chin-Chih Chen;Fen-Lang Chiang;Chun-Huey Huang

doi:10.6156/JTAR.201809_67(3).0001

宮燈百合Gloriosa stripe mosaic virus 之核酸分子和免疫法鑑定

Other Title

Molecular and Serological Identification of Gloriosa stripe mosaic virus on Christmas Bells (Sandersonia aurantiaca Hook)

Journal

台灣農業研究

Journal Volume

67

Journal Issue

3

Start Page

229

End Page

246

ISSN

0022-4847

Date Issued

2018-09-01

Date

2018-09-01

Author(s)

陳金枝

江芬蘭

黃春惠

Chin-Chih Chen

Fen-Lang Chiang

Chun-Huey Huang

DOI

10.6156/JTAR.201809_67(3).0001

URI

https://scholars.tari.gov.tw/handle/123456789/14185

Abstract

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 (coat protein; 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。 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.

Subjects

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

Publisher

農業試驗所

Type

journal article

File(s)

Name

67-3-1.pdf

Size

4.24 MB

Format

Adobe PDF

Checksum

(MD5):b9197f481804b321bc0ec049afe1768f