臺灣博碩士論文加值系統

自南投香草苗圃購得的水果鼠尾草盆栽，葉子有明顯的嵌紋病徵，將病葉經奎藜三次單斑分離所得之病毒機械接種於15科37種植物，結果計有藜科植物奎藜、紅藜及菠菜等3種及莧科植物千日紅1種可經機械接種感染。純化病毒懸浮液以2%醋酸鈾陰染，於電子顯微鏡可觀察到直徑約26 nm之球形病毒。病毒之物理性質包括熱不活化溫度介於55-60 ℃、耐稀釋度為10-3-10-4、室溫下耐保存性為8天。利用高低速交互離心及硫酸銫密度梯度離心純化罹病奎藜病葉時，分別在距離心管液面4.8 cm與5.7 cm處，出現白色病毒帶。光度吸收曲線圖方面，其Amax在258 nm，Amin在237 nm，Amax/Amin=1.62，A260/A280為1.73，純化100 g奎藜病葉可得2.08 mg病毒。將此病毒與13種病毒抗血清，進行間接和直接酵素結合抗體反應測試，顯示只有對蠶豆萎凋病毒2 (Broad bean wilt virus 2, BBWV-2)抗血清有反應。純化病毒以SDS-PAGE凝膠電泳分析病毒鞘蛋白，得到兩種蛋白次單位，分子量分別是21及43 kDa。利用專一引子對經RT-PCR將部份RNA 2核酸選殖、譯讀，得到長度1833 個核苷酸，包含大鞘蛋白及小鞘蛋白基因。大鞘蛋白(LCP)基因序列為1206個核苷酸，其所對應轉譯蛋白質可產生402個胺基酸，分子量經估算約為44.3 kDa。小鞘蛋白(SCP)基因序列為594個核苷酸，其所對應轉譯蛋白質可產生197個胺基酸，分子量約為22.2 kDa。利用DNASIS及VECTOR NTI 軟體與NCBI資料庫的蠶豆萎凋病毒的進行LCP與SCP核苷酸序列及胺基酸序列比對。核苷酸序列比對結果與屬於BBWV-2的Patchouli mild mosaic virus (accession number AB11007)的相同度最高，LCP及SCP均為95%；而與BBWV-1的PV176品系(accession number AB018703) 相同度最低，LCP為61%，而SCP為60%。胺基酸序列比對結果，與屬於BBWV-2的MB7分離株(accession number AB013616)相似度最高，LCP為98%而SCP為97%；而與BBWV-1的PV131品系(accession number AB018702) 相似度最低，LCP為64%及SCP僅59%。由親源圖顯示，本病毒為蠶豆萎凋病毒2的一個品系(strain)，初步命名為蠶豆萎凋病毒水果鼠尾草品系(BBWV2-FS)；此為台灣水果鼠尾草病毒病害之首度報導。

Fruit sage (Salvia dorisiana) seedlings with mosaic symptom were found from the herb nursery in Nantou. The virus isolate was obtained from three successive single lesion isolations on Chenopodium quinoa and subsequently tested for its infectivity on 37 plant species from 17 families by mechanical inoculation. Three species in Chenopodiaceae including Chenopodium amaranticolor, C. quinoa and Spinacia oleracea and one of the plants from Amaranthaceae, Gomphrena globosa, could be infected. Examination of preparations of purified virus by electron microscopy revealed some spherical particles of 26 nm in diameter were observed. The thermal inactivation point of the virus was 55 ℃ to 65 ℃, dilution end-point was 10-3 to 10-4 and the longevity in vitro of the infectious sap was 8 days at room temperature. Differential centrifugation followed by cesium sulphate (Cs2SO4) density gradient centrifugation of preparations from tissues of infected C. quinoa were conducted. Two white bands of virus were observed below top surface 4.8 cm and 5.7 cm of the centrifuge tube after purification. The virus had a maximal absorption at 258 nm and a minimal absorption at 237 nm. Amax/Amin and A260/A280 ratio were 1.62 and 1.73, respectively. The yield of purification from 100 g tissue of C. quinoa were 2.08 mg. When thirteen kinds of antisera were used in direct or indirect ELISA, it only reacted to the antiserum to Broad bean wilt virus 2 (BBWV-2). Analysis by SDS-polyarylamide gel electrophoresis of purified virus preparations showed that the viron contained two structural polypeptides with 21 and 43 kDa relative molecular weights, respectively. Cloning and sequencing part of viral RNA 2 by RT-PCR (reverse transcription- polymerase chain reaction) with degenerate and specific primer pairs obtained 1833 nucleotides encoding a large coat protein (LCP) and a small coat protein (SCP). The LCP gene with 1206 nucleotides encoded a 44.3 kDa protein of 402 amino acids. The SCP gene with 594 nucleotides encoded a 22.2 kDa protein of 197 amino acids. The nucleotide and amino acid sequences of LCP and SCP genes were compared with BBWV sequences in NCBI database by DANSIS and VECTOR NTI software. The highest nucleotide identity of LCP and SCP genes were both 95% with Patchouli mild mosaic virus (accession number AB11007). The lowest identity of LCP and SCP genes were 61% and 60% with PV176 strain of BBWV-1 (accession number AB018703), respectively. The highest amino acid similarity of LCP and SCP genes were 98% and 97% with MB7 isolate of BBWV-2 (accession number AB013616), respectively. The lowest similarities of LCP and SCP genes were 64% and 59% with PV131 strain of BBWV-1 (accession number AB018702), respectively. Phylogenetic tree obtained from the alignments of the LCP and SCP genes amino acid sequences showed that the virus was a strain of BBWV-2 and named BBWV2-FS. This is the first report of natural occurrence of BBWV to cause a disease on fruit sage in Taiwan.

中文摘要……………………………………………….……….. Ⅰ
英文摘要…………………………………………….………….. Ⅲ
壹、前言………………………………………………………… 1
一、鼠尾草簡介……………………………………………….. 1
二、可感染鼠尾草的病毒種類……………………………….. 3
三、蠶豆病毒屬(Fabavirus)的簡介…………………………… 3
四、蠶豆萎凋病毒(Broad bean wilt virus)的特性簡介. 4
貳、材料與方法……………………………………………..….. 10
一、病毒之來源與分離………………………………….….... 10
二、寄主範圍之測定…………………………………………. 10
三、病毒之物理性質測定……………………………….…… 10
四、電子顯微鏡觀察…………………………………………. 11
五、病毒之純化………………………………………………. 11
六、與多種病毒抗血清進行indirect和direct ELISA測試 13
七、RNA萃取與電泳分析….………………..………………. 13
八、病毒鞘蛋白質分子量大小分析..…………..……………. 14
九、病毒鞘蛋白基因之定序..…………………..……………. 15
十、鞘蛋白基因序列之比較…………………………………. 18
十一、利用反轉錄聚合酶連鎖反應(reverse transcription-poly-
merase chain reaction)偵測水果鼠尾草蠶豆萎凋病毒. 19
參、結果………………………………………………………... 21
一、病毒之來源與分離………………………………………. 21
二、寄主範圍之測定…………………………………………. 21
三、病毒之物理性質測定……………………………………. 21
四、電子顯微鏡觀察…………………………………………. 31
五、病毒之純化………………………………………………. 31
六、與多種病毒抗血清進行indirect和direct ELISA測試 37
七、病毒RNA的萃取………………………………………… 37
八、病毒鞘蛋白質分子量大小分析……………………..…… 41
九、病毒鞘蛋白基因之定序………………………………….. 41
十、鞘蛋白基因序列之比較………………………………….. 48
十一、利用反轉錄聚合酶連鎖反應偵測水果鼠尾草蠶豆萎凋
病毒…………………………….………………………. 54
肆、討論………………………………………………………… 60
伍、參考文獻…………………………………………………… 66

王惠亮、陳滄海。2001。中國大陸重要經濟果樹蔬菜作物病毒病。行政院農業委員會動植物防疫檢疫局編印。153-155頁。
尤次雄。2002。香草生活家。台視文化公司。30-31頁。
周雪平、李德葆。1996。蠶豆萎蔫病毒純化和病毒蛋白質及RNA組份分析。病毒學報12：376-373。
周雪平、余永杰、戚益軍、陳正賢、李德葆。1996。蠶豆萎蔫病毒ELISA檢測系統建立及其應用。植物病理學報26：347-352。
周雪平、濮祖芹、方中达。1994。豌豆病毒病原研究。植物病理學報24：207-212。
陳紹崇、吳雅芳、彭瑞菊、鄭安秀。2004。香草植物病害。農業世界雜誌251：81-83。
陳慶忠、柯文華、曹淑麗、趙佳鴻。2001。洋桔梗上蠶豆萎凋病毒之分離與鑑定。台中區農業改良場研究彙報70：51-63。
陳慶忠、陳煜焜、柯文華、徐惠迪。2002。康乃馨上洋桔梗壞疽病毒之分離與鑑定。植物病理學會刊11：137-146。
張元聰、王仕賢、王裕權。2002。西洋香草介紹四－鼠尾草。農業世界雜誌224：85-89。
張定霖、吳昭祥、洪進雄。2003。香藥草植物圖鑑。行政院農委會種苗改良繁殖場編印。109頁。
張瑋哲。2005。活體外齒舌蘭輪斑病毒台灣系統轉錄體之構築與其5΄端鳥糞嘌呤序列數目對於病毒感染力之影響。國立高雄師範大學生物科學研究所碩士論文。24-25頁。

黃鵬錡。2004。香草種植手冊。台北市上旗文化。183-189頁。
盧幽枝。2005。由矮牽牛分離之胡瓜嵌紋病毒特性之研究。國立高雄師範大學生物科學研究所碩士論文。16-17、20頁。

Brunt, A. A., Crabtree, K., Dallwitz, M. J., Gibbs, A. J., Watson, L., and Zurcher, E. J. 1996. Plant Viruses Online: Description and Lists from the VIDE Database. Version: 20th August 1996.’ URL http://biology.anu.edu.au/Groups/MES/vide/
Büchen-Osmond, C. (Ed). 2006. ICTVdB Management. Fabavirus. In: ICTVdB - The Universal Virus Database, version 4, Columbia University, New York, USA.
Büchen-Osmond, C. (Ed). 2006. ICTVdB Management. Broad bean wilt virus 1 and 2. In: ICTVdB - The Universal Virus Database, version 4, Columbia University, New York, USA.
Doel, T. R. 1975. Comparative properties of type, Nasturtium ringspot and Petunia ringspot strains of Broad bean wilt virus. J. Gen. Virol. 26: 95-108.
Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U., and Ball, L. A. 2005. Virus Taxonomy: 8th Report of the International Committee on Taxonomy of Viruses. Elsevier Academic press, Oxford. pp 812-813.
Hudaib, M., Bellardi, M. G., Rubies-Autonell, C., Fiori, J., and Cavrini, V. 2001. Chromatographic (GC-MS, HPLC) and virological evaluations of Salvia sclarea infected by BBWV-1. Il Farmaco 56: 219-227.
Ikegami, M., Kawashima, H., Natsuki, T., and Sugimura, N. 1998. Complete nucleotide sequence of the genome organization of RNA 2 of Patchouli mild mosaic virus, a new Fabavirus. Arch. Virol. 143: 2431-2434.
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., Kamea-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.
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.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
Lee, U., Hong, J. S., Choi, J. K., Kim, K. C., Kim, Y. S., Curtis, I. S., Nam, H. G., and Lim, P. O. 2000. Broad bean wilt virus causes necrotic symptoms and generates defective RNAs in Capsicum annuum. Phytopathology 90: 1390-1395.
Mumford, R. A., Jarvis, B., Harju, V., Boonham, N., and Skelton, A. 2006. The first report of Broad bean wilt virus 2 in the UK: findings in foxglove and salvia. Plant Pathol. 55: 818-819.
Natsuaki, K. T., Tomaru, K., Ushiku, S., Ichikawa, Y., Sugimura, Y., Natsuaki, T, Okuda, S., and Teranaka, M. 1994. Chracterization of two viruses isolated from patchouli in Japan. Plant Dis. 78: 1094-1097.
Qi, Y. J., Zhou, X. P., and Li, D. B. 2000a. Complete nucleotide sequence and ifectious cDNA clone of the RNA1 of a Chinese isolate of Broad bean wilt virus 2. Virus Genes 20 (3): 201-207.
Qi, Y. J., Zhou, X. P., Tao X. R., and Li, D. B. 2000b. Characterization and RNA2 nucleotide sequence of Broad bean wilt virus 2 isolate P158. J. Zhejiang Uni. Sci. 1: 437-441.
Qi, Y. J., Zhou, X. P., Huang, X. Z., and Li, G. X. 2002. In vivo accumulation of Broad bean wilt virus 2 VP37 protein and its ability to bind single-stranded nucleic acid. Arch. Virol. 147: 917-928.
Sanger, F., Nicklen, S., and Coulson, A. R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74 (12): 5463-5467.
Sanz, N. T., Chen, T. H., and Lai, P. Y. 2001. A newly discovered mosaic disease of bush basil (Ocimum bacilicum) in Taiwan. Plant Pathol. Bull. 10: 155-164.
Shukla, D. D., and Gough, K. H. 1983. Tobacco streak, Broad bean wilt, Cucumber mosaic, and Alfalfa mosaic viruses associated with ring spot of Ajuga reptans in Australia. Plant Dis. 67: 221-224.
Taylor, R. H., Smith, P. R., Reinganum, C., and Gibbs, A. J. 1968. Purification and properties of Broad bean wilt virus. Aust. J. Biol. Sci. 21: 929-935.
Taylor, R. H., and Stubbs, L. L. 1972. Broad bean wilt virus. AAB Descriptions of Plant Viruses No. 81.
Uyemoto, J. K., and Provvidenti, R. 1974. Isolation and identification of two serotypes of Broad bean wilt virus. Phytopathology 64: 1547-1548.
Xu, Z. G., Cockbain, A. J., Woods, R. D., and Govier, D. A. 1988. The serological relationships and some other propertiesof isltes of Broad bean wilt virus from faba and pea in China. Ann. Appl. Biol. 113: 287-296.
Zhou, X. P. 2002. Broad bean wilt virus 2. AAB Descriptions of Plant Viruses No. 392.