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研究生:沈炳男
研究生(外文):Bing-Nan Shen
論文名稱:自台灣洋桔梗分離的蠶豆萎凋病毒-2之特性及其全基因組之選殖及分析
論文名稱(外文):Characterization, complete genome sequence and genetic organization of Broad bean wilt virus-2 isolated from lisianthus in Taiwan
指導教授:詹富智
指導教授(外文):Fuh-Jyh Jan
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
畢業學年度:96
語文別:英文
論文頁數:57
中文關鍵詞:洋桔梗蠶豆萎凋病毒-2
外文關鍵詞:lisianthusBroad bean wilt virus-2
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洋桔梗 [Eustoma grandiflorum (Ref.) Shinners] 是一種在全世界廣受歡迎的觀賞花卉植物。1996年,彰化地區栽種的洋桔梗葉片顯現出黃綠色環斑及壞疽病斑病徵,經過分離後得到一病毒分離株,命名為LV-5。在先前的報告中指出由於LV-5可與市售的BBWV-2抗血清反應,因此認為LV-5可能是屬於蠶豆萎凋病毒-2(Broad bean wilt virus-2, BBWV-2)的其中一個分離株。本次研究的目標在於以分子生物學方式提供資料,以確定LV-5病毒分離株為BBWV-2的成員之一,並且將此病毒的全基因組加以選殖定序、分析其生物特性、以及生產針對此病毒的多株抗體。將病毒自感染LV-5病毒分離株之菸草中純化,在電子顯微鏡底下可以觀察到大量直徑約28 nm的球型病毒顆粒。將兔子以純化的病毒進行免疫後,即可獲得多株抗體,並且以ELISA及Western blotting 的方式確認其偵測此病毒之能力。而寄主範圍反應測定結果顯示BBWV-2 LV-5分離株可以感染4個科共17種植物。LV-5的鞘蛋白全長序列經由反轉錄-聚合
Lisianthus [Eustoma grandiflorum (Ref.) Shinners] is a popular herbaceous ornamental plant worldwide. In 1996, a virus isolate LV-5 was obtained from lisianthus plants showing chlorotic and necrotic spots on leaves in Changhua, Taiwan. The virus was previously identified as a potential isolate of Broad bean wilt virus-2 (BBWV-2) since it positively reacted with a commercial polyclonal antibody against BBWV-2. The objectives of this study were to provide the molecular evidence to confirm that the virus isolate LV-5 is indeed a BBWV-2, to clone and sequence the genome of this virus, to characterize the biological properties and generate polyclonal antibody against this virus. Virus particles were purified from infected Nicotiana benthamiana plants and 28 nm isometric virions were observed by an electron microscopy. Polyclonal antibody was produced by immunizing the rabbit with purified virus, and the ability to detect BBWV-2 isolate LV-5 was demonstrated by western blotting and ELISA. The host range reactions test showed that 17 plant species belonging to 4 families were susceptible to BBWV-2 isolate LV-5. Coat protein (CP) genes of LV-5 were amplified by RT-PCR and then cloned and sequenced. Comparisons of the nucleotide sequence of large-CP (L-CP) and small-CP (S-CP) showed that LV-5 shared 77.6-92.3% and 77.1-93.9% identities, respectively, to those of other eighteen BBWV-2 isolates. Comparisons of the amino acid sequence of L-CP and S-CP showed that LV-5 shared 84.8-97.5% and 88.8-98.0% identities, respectively, to those of other BBWV-2 isolates. The L-CP of LV-5 has 61.5-62.3% nucleotide and 62.7-64.4% amino acid identies with those of BBWV-1 isolates. The S-CP of LV-5 has 60.5-62.4% nucleotide and 58.4-59.9% amino acid identities with those of BBWV-1 isolates. Near full-length genomic sequences was obtained via genome walking techniques and the 5´ terminal sequences were obtained from RT-PCR with oligo-d(T) as primers using polyadenylated dsRNA as template. Excluding the 3´ terminal poly-A tails, the RNA 1 and 2 are 5947 nt and 3555 nt, respectively. All genes on both RNAs were determined according to the proposed protease cleavage sites. Comparison of the full-length sequences showed that the LV-5 isolate shared 78.6-93.0% identity on RNA 1 and 78.7-92.0% on RNA 2 to those of other seven published BBWV-2 isolates. Phylogenetic analyses based on the full-length nucleotide sequences of RNA 1 or RNA 2 and the amino acid sequences of two coat proteins showed that LV-5 isolated from lisianthus in Taiwan was closely related to the BBWV-2 IP isolate infecting pepper in Japan. Taken together, our results provide the molecular evidence to confirm that virus isolate LV-5 from lisianthus in Taiwan is an isolate of BBWV-2.
Abstract in Chinese ....................................................................................1
Abstract in English .....................................................................................3
Literature review ........................................................................................5
Introduction and literature review .........................................................12
Materials and Methods ............................................................................19
Virus source and maintenance ................................................................................19
Indirect ELISA tests.................................................................................................19
Western blotting ......................................................................................................20
Purification of virus particles ..................................................................................21
Electron microscopy................................................................................................22
Production of polyclonal antibody ..........................................................................22
Double immuno-diffusion test ................................................................................23
Host-range reactions ...............................................................................................23
Total RNA extraction ..............................................................................................25
Design of the degenerate primer pairs for CP and 3´-NCR region cloning of
BBWV-2 and BBWV-1 ...........................................................................................25
Reverse transcriptase-polymerase chain reaction (RT-PCR) ..................................26
cDNA cloning, sequencing, assembly, and computer analysis ...............................27
Phylogenic analyses of the coat protein gene of BBWV-2 isolate LV-5 ................28
Design of the degenerate primer pairs of BBWV-2 ................................................29
Viral double-strand RNA (dsRNA) extraction ........................................................29
Polyadenylation of BBWV-2 dsRNA .....................................................................30
Cloning of the 5´ terminal of LV-5 genome ............................................................31
Full-length sequence comparison between LV-5 with other BBWV-2
Isolates ....................................................................................................................31
Results ........................................................................................................33
Virus source and maintenance ................................................................................33
Properties of purified virus .....................................................................................33
SDS double immuno-diffusion test .........................................................................34
Western blotting analyses .......................................................................................34
Serial dilution test of polyclonal antibody ..............................................................35
Host-range reactions ...............................................................................................35
RT-PCR detection for the CP region .......................................................................36
Comparison of the nucleotide and amino acid sequences of L-CP and
S-CP of LV-5 with those of other BBWV isolates ..................................................36
Cloning and sequencing of the near full-length RNA 1of BBWV-2 ......................37
Isolation of viral dsRNA .........................................................................................37
Cloning of the 5´ terminal of BBWV-2 isolate LV-5 genome ................................38
Complete genome sequence of BBWV-2 isolate LV-5 ...........................................38
Assignment of the cleavage sites of the viral polyprotein ......................................39
Comparison of the nucleotide and amino acid sequences of BBWV-2
isolate LV-5 with other isolates ...............................................................................39
Discussion ..................................................................................................41
References ..................................................................................................48
Tables and Figures ....................................................................................58
鄭櫻慧、張清安。2003。雙生病毒。植物保護圖鑑系列-洋桔梗保護,100-103頁。行政院農委會動植物防疫檢疫局出版。台北,118頁。
鄭櫻慧、陳金枝、王如玉、張清安。2007。台灣粉蝨傳播感染花卉作物之雙生病毒,269-278頁。2007植物蟲媒病害與防治研討會專刊。行政院農委會動植物防疫檢疫局出版。台北,278頁。
陳慶忠。2003。蠶豆萎凋病毒。植物保護圖鑑系列-洋桔梗保護,87-89頁。行政院農委會動植物防疫檢疫局出版。台北,118頁。
王裕權、張元聰、王仕賢。2003。前言。植物保護圖鑑系列-洋桔梗保護,1-5頁。行政院農委會動植物防疫檢疫局出版。台北,118頁。
Chang, C. A., and Tsai, H. T. 1993. Isolation of Bean yellow mosaic virus from lisianthus developing foliar mosaic and flower breaking symptoms. Plant Pathol. Bull. 2: 250-251.
Chao, C. H., Chen, C. C., Chang, C. A., and Chen, C. C. 2000. Identification of a Turnip mosaic virus isolate causing systemic yellow spotting on lisianthus. Plant Pathol. Bull. 9: 115-122.
Chen, C. C., Chen, Y. K., and Hsu, H. T. 2000. Characterization of a virus infecting lisianthus. Plant Dis. 84: 506-509.
Chen, C. C., and Hsu, H. T. 2002a. Occurrence of a severe strain of Lisianthus necrosis virus in imported carnation seedlings in Taiwan. Plant Dis. 86: 444.
Chen, C. C., and Hu, C. C. 1999. Purification and characterization of a cucumovirus from Lisianthus rusellianus. Plant Prot. Bull. 41: 179-198.
Chen, C. C., Hu, C. C., Chen, Y. K., and Hsu, H. T. 2002b. A fabavirus inducing ringspot disease in lisianthus. Acta Hortic. 568: 51-57.
Chen, C. C., Hu, C. C., and Hsu, H. T. 2001a. Characterization of a fabavirus isolated from diseased lisianthus. Phytopathology 91: S17.
Chen, C. C., Ko, W. F., and Chen, Y. K. 1998. Identification and transmission of a cucumovirus from Lisianthus rusellianus. Bull. Taichung Dist. Agri. Improv. Stn. 60: 1-18.
Chen, C. C., Ko, W. F., Tsao, S. L., and Chao, C. H. 2001b. Isolation and identification of a Fabavirus from lisianthus. Bull. Taichung Dist. Agri. Improv. Stn. 70: 51-63.
Chen, C. C., Tsao, S. L., and Hsu, H. T. 2001c. Diagnosis of Lisianthus necrosis virus infection by light and electron microscopy and serological assay. Plant Pathol. Bull. 10: 105-114.
Christie, S. R., Purcifull, D. E., Carawford, W. E., and Ahmed, N. A. 1987. Electron microscopy of negatively stained clarified viral concentrates obtained from small tissue samples with appendices on negative staining techniques. Fla. Agric. Exp. Stn. Bull. 872: 45.
Chu, F. H., Chao, C. H., Chung, M. H., Chen, C. C., and Yeh, S. D. 2001. Completion of the genome sequence of Watermelon silver mottle virus and utilization of degenerate peimers for decting tospoviruses in five serogeoups. Phytopathology 91: 361-368.
Clark, M. F., and Adams, A. N. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34: 475-483.
Cohen, J., Gera, A., Ecker, R., Ben, J. R., Perlsman, M., Gokkes, M., Lachman, O., and Antignus, Y. 1995. Lisianthus leaf curl a new disease of lisianthus caused by Tomato yellow leaf curl virus. Plant Dis. 79: 416-420.
Cohen, J., Lapidot, M., Loebenstein, G., and Gera, A. 2001. First report of Sweet potato sunken vein virus occurring in lisianthus. Plant Dis. 85: 679.
de Freitas, J. C., Kitajima, E. W., and Rezende, J. A. M. 1996. First report of Tobacco streak virus on lisianthus in Brazil. Plant Dis. 80: 1080.
Dikova, B. 2005. Tobacco rattle virus (TRV) transmission by sugar beet seeds. Biotechnol. Biotechnol. Equip. 19: 80-86.
Doi, M., Zen, S., Okuda, M., Nakamura, H., Kato, K., and Hanada, K. 2003. Leaf necrosis disease of lisianthus (Eustoma grandiflorum) caused by Iris yellow spot virus. Ann. Phytopathol. Soc. Jpn. 69: 181-188.
Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.
Ferrer, R. M., Guerri, J., Luis-Arteaga, M. S., Moreno, P., and Rubio, L. 2005. The complete sequence of a Spanish isolate of Broad bean wilt virus 1 (BBWV-1) reveals a high variability and conserved motifs in the genus Fabavirus. Arch. Virol. 150: 2109-2116.
Ferrer, R. M., Luis-Arteaga, M., Guerri, J., Moreno, P., and Rubio, L. 2007. Detection and identification of species of the genus Fabavirus by RT-PCR with a single pair of primers. J. Virol. Methods 144: 156-160.
Freitas, J. C., de Pozzer, L., Rezende, J. A. M., Kitajima, E. W., Avila, A. C., and de Resende, R. O. 1997. Lisianthus: A new ornamental affected by tomato spotted wilt virus in Brazil. Fitopatologia Brasileira. 22: 114.
Frowd, J. A., and Tomlinson, J. A. 1972. Relationships between a parsley virus, nasturtium ringspot virus and broad bean wilt virus. Ann. Appl. Biol. 70: 189-195.
Fujinaga, M., Morikawa, T., Doi, M., Yoneyama, C., Ibrahim, M., Ogiso, H., Miyamoto, K., Miyasaka, M., Ohki, T., Kameya-Iwaki, M., and Natsuaki, T. 2006. Two tombusviruses isolated from lisianthus [Eustoma grandiflorum (Raf.) Shinn.] with necrotic stunt. Jpn. J. Phytopathol. 72: 109-115.
Gera, A. G., and Cohen, J. 1990. The nature occurance of Bean yellow mosaic, Cucumber mosaic and Tobacco mosaic virus in lisianthus in Israel. Plant Pathol. 39: 561-564.
Goldbach, R., Martelli, G. P., and Milne, R. G. 1995. Family Comoviridae. p.341-347 in: Murphy, F. A., Fauquet, C. M., Bishop, D. H. L., Ghabrial S. A., Jarvis, A. W., Martelli, G. P., and Mayo, M. A., and Summer M. D. (eds.), Virus Taxonomy: Classification and Nomenclature of Viruses. Sixth report of the international committee on taxonomy of viruses. Springer, Wien New York, USA.
Gooderham, K. 1984. Transfer techniques in protein blotting. p.165-178 in: Walk, J. M. (ed.), Molecular Biology. Humana Press, Clifton, NJ.
Honda, Y., Omura, T., Kobayashi, Y. O., and Mikoshiba, Y. 2004. Detection of Broad bean wilt virus 1 and Broad bean wilt virus 2 in Japan. Ann. Rep. Kanto-Tosan Plant Prot. Soc. 51: 43-48.
Huang, K. L. 2005. Lisianthus. p.699-704 in: Yeh, Y. and Lin, T. C. (eds.), Taiwan agriculture encyclopedia (Crop edition-2). Harvest farm magazine, Taipei, Taiwan.
Ikegami, M., Kawashima, H., Natsuaki, T., and Sugimura, N. 1998. Complete nucleotide sequence of the genome organization of RNA 2 of patchouli mild mosaic virus, a new favavirus. Arch. Virol. 143: 2431-2434.
Iwaki, M., Hanada, K., Maria, E. R. A., and Onogi, S. 1987. Lisianthus necrotic virus, a new necrovirus from Eustoma russellianum. Phytopathology 77: 867-870.
Iwaki, M., Maria, E. R. A., Hanada, K., Onogi, S., and Zenbayashi, R. 1985. Three viruses occurred in lisianthus plants. Ann. Phytopathol. Soc. Jpn. 51: 355.
Jan, F. J. 1998. Roles of nontarget DNA and viral gene length in influencing multi-virus resistance through homology-dependent gene silencing. ph. D. Dissertation, Department of Plant Pathology, Cornell University.
Jan, F. J., Chen, C. C., and Hsu, H. T. 2003a. Identification of Tomato mosaic virus infection in lisianthus in Taiwan. Plant Dis. 87: 1537.
Jan, F. J., and Yeh, S. D. 1995. Purification, in situ localization, and comparative serological properties of passionfruit woodiness virus-encoded amorphous inclusion protein and two other viral proteins. Phytopathology 85: 64-71.
Jan, F. J., Zheng, Y. X., Chao, C. H., Ko, W. F., Chang, C. A., and Chen, C. C. 2003b. Identification of a tobamovirus causing yellow mottle and stunting symptoms on lisianthus in Taiwan. Plant Pathol. Bull. 12: 122-132.
Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120.
Kobayashi, Y. O., Kobayashi, A., Hagiwara, K., Uga, H., Mikoshiba, Y., Naito, T., Honda, Y., and Omura, T. 2005. Gentian mosaic virus: A New Species in the Genus Fabavirus. Phytopathology 95: 192-197.
Kobayashi, Y. O., Nakano, M., Kashiwazaki, S., Naito, T., Mikoshiba, Y., Shiota, A., Kameya-Iwaki, M., and Honda, Y. 1999. Sequence analysis of RNA-2 of different isolates of broad bean wilt virus confirms the existence of two distinct species. Arch. Virol. 144: 1429-1438.
Koh, L. H., Cooper, J. I., and Wong, S. M. 2001. Complete sequences and phylogenetic analyses of a Singapore isolate of broad bean wilt fabavirus. Arch. Virol. 146: 135-147.
Kondo, T., Fuji, S. I., Yamashita, K., Kang, D. K., and Chang, M. U. 2005. Broad bean wilt virus 2 in yams. J. Gen. Plant Pathol. 71: 441-443.
Kritzman, A., Beckelman, S., Alexandrov, S., Cohen, J., and Lampel, M. 2000. Lisianthus leaf necrosis: A new disease of lisianthus caused by Iris yellow spot virus. Plant Dis. 84: 1185-1189.
Kuroda, T., Okumura, A., Takeda, I., Miura, Y., and Suzuki, K. 2000. Nucleotide sequence and synthesis of infectious RNA from cloned cDNA of broad bean wilt virus 2 RNA 2. Arch. Virol. 145: 787-793.
Kuroda, T., and Suzuki, K. 2003. Development and partial characterization of monoclonal antibodies against Broad bean wilt virus 2 22-kDa coat protein. Ann. Phytopathol. Soc. Jpn. 69: 403-406.
Le Gall, O., Iwanami, T., Karasev, A. V., Jones, T., Lehto, K., Sanfa
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