臺灣博碩士論文加值系統

2011年五月，南投縣信義鄉甜椒 (Capsicum annuum L.) 作物發生疑似由病毒引起之病害，病徵為葉片褪綠、頂芽捲曲且果實黃化斑駁，利用感染茄科作物之馬鈴薯Y病毒屬 (Potyvirus)、番茄斑萎病毒屬 (Tospovirus) 及菸草嵌紋病毒屬 (Tobamovirus) 簡併式引子對 (degenerate primers) 和胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV) 專一性引子對 (specific primers) 進行反轉錄聚合酶連鎖反應 (Reverse transcription-polymerase chain reaction, RT-PCR)，在使用廣效性番茄斑萎病毒屬部分複製酶基因 (L gene) 之簡併式引子對 (gL3637、gL4435c) 可增幅出約900 bp 之片段，經過選殖定序後確認甜椒病株受番茄斑萎病毒屬之病毒感染，將其以奎藜 (Chenopodium quinoa) 進行單斑分離，得病毒分離株暫命名為TwPep3。本研究之目的為利用全長度S RNA之選殖定序、病毒顆粒型態、生物學特性及血清學特性進行此病毒之分類鑑定，並製備可用以偵測之多元抗血清 (polyclonal antiserum)。利用超薄切片法觀察TwPep3感染之圓葉菸草 (Nicotiana benthamiana)，可知TwPep3病毒顆粒型態為球型，直徑為80-110 nm。本研究藉由比對多種番茄斑萎病毒屬病毒設計S RNA簡併式引子對，以及配合聚腺苷酸化 (polyadenylated) 之雙股核醣核酸 (double-stranded RNA, dsRNA) 為模板，利用已知病毒序列設計專一性引子搭配oligo d(T) 引子進行RT-PCR增幅，完成全長度S RNA序列之定序。核鞘蛋白 (nucleocapsid protein) 基因長度為846-nt，預估分子量大小為31 kDa。將核鞘蛋白胺基酸序列與其他種之番茄斑萎病毒屬病毒比較，胺基酸相同度 (amino acid identity) 介於 19.1-89.0%；而在非結構蛋白S (non-structural protein S, NSs) 方面，胺基酸相同度為17.6-78.7%。與TwPep3同源性最高的病毒為番茄壞疽輪斑病毒 (Tomato necrotic ringspot virus, TNRV)。以純化之核鞘蛋白免疫紐西蘭白兔製備多元抗血清，利用西方轉漬法 (western blot) 分析感染TwPep3之植物組織，確認核鞘蛋白大小為31 kDa。寄主範圍共測試6科22種植物，其中茄科作物最為感病，多為系統性寄主，但TwPep3只會在番茄上形成局部單班。將TwPep3回接至甜椒上，與田間病株具有相似之葉片褪綠、頂芽捲曲之病徵，經所製備的抗血清檢測後確認TwPep3為引起前述田間甜椒病害之病原。依據TwPep3核鞘基因序列設計專一性引子對 (FJJ2012-67、FJJ2012-68) 進行田間調查，本病害目前在信義鄉發生，但仍可能有擴大之趨勢。根據核鞘蛋白之胺基酸相同度與已發表之番茄斑萎病毒屬病毒比較結果小於90%，以及血清學和生物學上之證據，確認TwPep3與TNRV有所差異，故認定為一種新的番茄斑萎病毒屬病毒，因此給予命名為Pepper chlorotic spot virus (PCSV)。

In May 2011, bell peppers displaying viral disease-like symptoms of chlorosis on leaves and deformation and yellow mottle on fruits were collected from Sinyi, Nantou County. By using a reverse-transcription polymerase chain reaction (RT-PCR) with the genus degenerated primers of pepper-infecting viruses, the partial RNA-dependent RNA polymerase gene (L gene) of tospoviruses was amplified. Based on the BLAST results of the 924-nucleotides partial L gene in GenBank, the causal agent on bell peppers was identified as a tospovirus. A virus culture designated as “TwPep3” was isolated from the symptomatic leaves. The virions of TwPep3 observed in ultrathin sections of symptomatic tissues with a transmission electron microscope showed isomertic particles with sizes of 80-110 nm in diameter. The full length sequence of TwPep3 S RNA was obtained by the stragery of genome walking. The degenerate primers for RT-PCR were designed for amplification of TwPep3 RNAs based on the analysis of the genomic sequences of the closely related tospoviruses. The sequences of 5’ and 3’ terminal regions of RNAs were amplified from polyadehylated double stranded RNAs with oligo d(T) primer. The comparison of the 846-nucleotides nucleocapsid (N) gene with those of the tospoviruses available in GenBank showed they shared 46.2-82.9% nucleotide identity and 19.1-89.0% amino acid identity. In addition, the amino acid identities of non-structural protein S (NSs) revealed 17.6-78.7%. Tomato necrotic ringspot virus (TNRV) was found to be the closest tospovirus to TwPep3. Polyclonal antiserum against TwPep3 was generated by immunizing rabbits with the purified 31-kDa N protein from TwPep3-infected Chenopodium quinoa. The host range study was determined by introducing TwPep3 to twenty-two plant species in six families. Systemic symptoms were observed on most of the tested plants in the family of Solanaceae, which were considered susceptible to TwPep3. The tissue extracts of back-inoculated bell pepper plants exhibiting similar symptoms to those observed on field peppers reacted positively to the antiserum against TwPep3 N protein. The specific primers designed from the viral sequences of TwPep3 N gene (FJJ2012-67 and FJJ2012-68) could be applied for virus survey in the fields. Based on the less than 90% amino acid identity of N protein when compared with those of other tospoviruses and the serological and biological characterization, virus TwPep3 is reported as a new tospovirus infecting bell peppers in Taiwan and designated as Pepper chlorotic spot virus (PCSV).

目次
中文摘要 i
Abstracts iii
目次 v
表目次 vii
圖目次 viii
一、 前言與前人研究 1
二、 材料與方法 5
(一) 病毒來源、分離、繁殖與保存 5
(二) 電子顯微鏡觀察 5
(三) 總量核醣核酸 (total RNA) 之抽取 6
(四) 簡併式 (degenerate) 與專一性 (specific) 引子對之設計 6
(五) 反轉錄-聚合酶連鎖反應 (Reverse transcription-polymerase chain reaction, RT-PCR) 7
(六) 病毒雙股核醣核酸 (dsRNA) 之抽取 8
(七) 雙股核醣核酸之聚腺苷酸化 (polyadenylation) 9
(八) 病毒3’ 及5’ 端序列之增幅 9
(九) 病毒基因之選殖及序列分析 10
(十) 病毒基因演化樹分析 10
(十一) 純化病毒核鞘蛋白 11
(十二) 多元抗體之製備 12
(十三) SDS免疫雙向擴散反應 (SDS-immuno double diffusion) 12
(十四) 西方轉漬法 (western blotting) 12
(十五) 酵素連結免疫吸附反應 (enzyme-linked immunosorbent assay, ELISA) 13
(十六) 回接及寄主範圍測試 14
(十七) 田間病害之調查 14
三、 結果 15
(一) 可疑病毒病害之檢測 15
(二) 電子顯微鏡觀察 15
(三) 全長度S RNA序列選殖定序 15
(四) 核鞘蛋白胺基酸序列之演化樹分析 16
(五) 多元抗血清之製備及其力價與專一性測試 16
(六) TwPep3與WSMoV血清群的關係 17
(七) 回接及寄主範圍測試 17
(八) 田間病害之調查 18
四、 討論 19
五、 參考文獻 23
圖與表 32

表目次
表一、本研究所使用之引子對 32
表二、甜椒黃化斑點病毒與其他番茄斑萎病毒之核鞘蛋白基因序列比對 33
表三、甜椒黃化斑點病毒與其他番茄斑萎病毒之NSs基因序列比對 35
表四、機械接種甜椒黃化斑點病毒至指示植物之寄主範圍測試結果 36

圖目次
圖一、甜椒黃化斑點病於甜椒之病徵 37
圖二、利用常見感染甜椒之病毒簡併式或專一性引子對進行甜椒黃化斑點病毒之反轉錄-聚合酶連鎖反應 (Reverse transcription-polymerase chain reaction, RT-PCR) 分析 38
圖三、利用電子顯微鏡觀察感染甜椒黃化斑點病毒之圓葉菸草葉片超薄切片中的病毒顆粒型態 39
圖四、甜椒黃化斑點病毒之S RNA基因體序列選殖策略 40
圖五、甜椒黃化斑點病毒與其他已發表之25種tospovirus之核鞘蛋白演化樹分析 41
圖六、利用SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 分析純化之甜椒黃化斑點病毒核鞘蛋白 42
圖七、利用雙向免疫擴散反應分析甜椒黃化斑點病毒之抗血清力價 43
圖八、利用甜椒黃化斑點病毒、番椒黃化病毒及西瓜銀斑病毒之多元抗血清以西方轉漬法分析三者之血清學關係 44
圖九、以西方轉漬法分析甜椒黃化斑點病毒核鞘蛋白分子量大小 45
圖十、甜椒黃化斑點病毒核鞘蛋白基因引子對之專一性測試及實際應用於田間檢測之情形 46

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