西瓜頂芽壞疽病毒基因體定序及利用簡併性引子對快速鑑定番茄斑萎病毒屬病毒

Determination of genomic sequence of Watermelon bud necrosis virus and quick identification of tospoviruses using degenerate primer pairs

葉怡均(Yeh Yi-Chun)

指導教授 ： 陳宗祺

亞洲大學/健康學院/生物科技學系碩士班/碩士(2008年)

摘要

番茄斑萎病毒屬 (the genus Tospovirus) 為布尼亞病毒科 (Bunyaviridae) 中唯一感染植物之一屬，其基因體由三條單股RNA片段所組成，依其大小分別命名為S、M、L，可分別轉譯出六種蛋白產物。Tospovirus之分類即根據S RNA上所轉譯出來的核鞘蛋白 (nucleocapsid protein，NP) 同源性 (homology) 來決定的。目前為止，本屬共發現有十九種 (species) 不同的病毒。然而，除了NP的序列已完全解出之外，其他病毒產物的基因體轉譯序列並未完全定序。本研究將完整呈現西瓜頂芽壞疽病毒 (Watermelon bud necrosis virus，WBNV) 的全基因體序列。WBNV的S RNA由3,402個核苷酸組成，為雙極性片段，病毒股RNA對應轉譯出含439個胺基酸的非結構性NSs蛋白，其互補股RNA產生275個胺基酸的NP。M RNA含有4,794個核苷酸亦為雙極性片段，病毒股對應產生307個胺基酸的非結構性NSm蛋白，而互補股則對應產生1,121個胺基酸的Gn和Gc醣蛋白的前驅物。L RNA共具8,914個核苷酸，為負極性片段，互補股對應產生2,878個胺基酸的RNA依賴性RNA聚合酶 (RNA-dependent RNA polymerase，RdRp)。序列分析結果顯示WBNV和花生頂芽壞疽病毒 (Peanut bud necrosis virus，PBNV)、西瓜銀斑病毒 (Watermelon silver mottle virus，WSMoV)、和甜椒黃化病毒 (Capsicum chlorosis virus，CaCV) 的基因體序列展現高度相似性。另一方面，從已知的病毒序列中設計新的簡併性引子對 (degenerate primer pairs)，並和已發表的簡併性引子對同時進行病毒檢測鑑定。本論文中針對下列十三種病毒：WBNV、PBNV、WSMoV、CaCV、番茄斑點萎凋病毒 (Tomato spotted wilt virus，TSWV)、花生輪點病毒 (Groundnut ringspot virus，GRSV)、番茄黃化斑點病毒 (Tomato chlorotic spot virus，TCSV)、鳳仙花壞疽斑點病毒 (Impatiens necrotic spot virus，INSV)、彩色海芋黃斑病毒 (Calla lily chlorotic spot virus，CCSV)、鳶尾花黃斑病毒 (Iris yellow spot virus，IYSV)、甜瓜黃斑病毒 (Melon yellow spot virus，MYSV)、花生黃化扇斑病毒 (Peanut chlorotic fan-spot virus，PCFV)、以及番茄黃化輪點病毒 (Tomato yellow ring virus，TYRV)，利用反轉錄聚合酶連鎖反應 (reverse transcription-polymerase chain reaction，RT-PCR) 加以測試。使用gL3637/gL4510c或M1/tNSm400c均可測得所有測試的病毒。若使用tNSm410/tNSm870或t2740/t3920，則除PCFV外，上述十二種病毒均可檢測出來。如果想檢測WSMoV和IYSV血清群病毒 (WSMoV、CaCV、CCSV、MYSV、WBNV、PBNV、IYSV、TYRV)，則可使用WG1/WG960或Gp2150/Gp2730c。在本研究中我們發展出利用簡併性引子對配合One-Step RT-PCR快速檢測tospoviruses的方法，此法亦可應用在解開未知tospoviruses的序列中。

關鍵字

番茄斑萎病毒屬 ； 西瓜頂芽壞疽病毒 ； 簡併性引子對

並列摘要

The genus Tospovirus of the family Bunyaviridae has a large genome that consists of three RNA segments, denoted S, M and L RNAs, to code six gene products. The homology of nucleocapsid protein (NP), encoded from the S RNA, is a key criterion for classification of Tospovirus. Based on the NP, 19 virus species within this genus have been characterized. However, except the NP, other coding sequences are still incompletely determined in Tospovirus. In this investigation, the entire genomic sequence of Watermelon bud necrosis virus (WBNV) was determined. The S RNA of WBNV is 3,402 nucleotides (nts) in length, encoding 439 amino acids (aa) of NSs protein in the viral (v) sense and a 275 aa of NP in the viral complementary (vc) strand. The M RNA is 4,794 nts in length encoding a 307aa of NSm protein in the v strand and a 1,121 aa of precursor of Gn and Gc glycoproteins (GPs) in the vc strand. The L RNA has 8,914 nts, is of negative polarity, that codes a 2,878 aa of RNA-dependent RNA polymerase in the vc sense. Sequence analyses revealed that WBNV is closely related to Peanut bud necrosis virus (PBNV), Watermelon silver mottle virus (WSMoV) and Capsicum chlorosis virus (CaCV). On the other hand, degenerate primer pairs were designed from the comparison of the determined tospoviral sequences. The previously reported degenerate primer pairs were also used for virus detection in this study. Thirteen distinct tospovirus species, WBNV, PBNV, WSMoV, CaCV, Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Tomato spotted wilt virus (TSWV), Tomato chlorotic spot virus (TCSV), Calla lily chlorotic spot virus (CCSV), Iris yellow spot virus (IYSV), Melon yellow spot virus (MYSV), Peanut chlorotic fan-spot virus (PCFV) and Tomato yellow ring virus (TYRV), were tested by reverse transcription-polymerase chain reaction (RT-PCR). All analyzed tospoviruses can be detected by primer pairs gLl3637/gL4510c and M1/tNSm400c. The primer pairs tNSm410/tNSm870 and t2740/t3920 can be used to detect 12 tospoviruses, except PCFV. WSMoV, CaCV, CCSV, MYSV, WBNV, PBNV, IYSV and TYRV, belonging to both WSMoV and IYSV serogroups, can be detected by the primer pairs WG1/WG960 and Gp2150/Gp2730c. Here, we developed a quick method for detection of tospoviruses with RT-PCR using degenerate primer pairs. Moreover, this method can be applied for determining unknown tospoviral sequences.

並列關鍵字

Tospovirus ； Watermelon bud necrosis virus ； degenerate primer pairs

參考文獻

Department of Plant Pathology, National Chung Hsing University.

1. Amin, P. W., Reddy, D. V. R., and Ghanekar, A. M. 1981. Transmission of Tomato spotted wilt virus, causal agent of bud necrosis virus of peanut, by Scirthothrips dorsalis and Frankliniella schultzei. Plant Dis. 65:663-665.

3. Bag, S., Druffel, K. L., Salewsky, T., and Pappu, H. R. 2009. Nucleotide sequence and genome organization of the mediumRNA of Iris yellow spot virus from the United States. Arch. Virol. 154:715-718.

4. Best, R. J. 1968. Tomato spotted wilt virus. Adv. Virus Res. 13:65-145.

5. Bezerra, I. C., Resende, R. O., Pozzer, L., Nagata, T., Kormelink, R., and de Avila, A. C. 1999. Increase of tospoviral diversity in Brazil with the identification of two new tospovirus species, one from chrysanthemum and one from zucchini. Phytopathology 89:823-830.