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研究生:何宇倫
研究生(外文):Yu-Lun Ho
論文名稱:茄科重要病毒鑑定晶片之研發與應用
論文名稱(外文):Development of Identification Chip for Important Solanaceous Plant Viruses
指導教授:張雅君張雅君引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理與微生物學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:58
中文關鍵詞:生物晶片茄科作物馬鈴薯Y病毒屬菸草嵌紋病毒屬胡瓜嵌紋病毒屬
外文關鍵詞:MicroarraySolanaceous plantsPotyvirusTobamovirusCucumovirus
相關次數:
  • 被引用被引用:4
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  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:1
茄科作物為重要的蔬菜和水果,生長期間常遭受病毒危害,且田間的植株可能會被一種以上的病毒複合感染。依據國外資料記載,可感染茄科作物的病毒種類超過20屬80種以上,而國內也有40種以上的病毒可以感染茄科作物,其中又以cucumoviruses、potyviruses和tobamoviruses最為嚴重,對作物造成重大損失。在面對這些可能遭受各種病毒感染的作物,常使用的檢測方法有ELISA、RT-PCR等,但為達成一次實驗可同時診斷多種病害的目標,我們將生物晶片應用於茄科病毒之鑑定,以cucumoviruses、potyviruses和tobamoviruses作為診斷鑑定晶片的檢測對象。我們的研究策略需要設計簡併性引子對(degenerate primer),因此先從GenBank收集相關病毒的胺基酸序列,再利用ClustalX程式進行胺基酸序列多序列並列比對,根據比對結果利用CODEHOP軟體找出具有保守性的區域,再依照該軟體的策略設計簡併性引子。之後以簡併性引子對所圍成的區域,找出病毒種間差異性較大的區段,設計專一性寡核苷酸探針(oligonucleotide probe)。請廠商合成50 mer寡核苷酸探針之後點漬在載體上,載體可為尼龍膜或是經過處理的玻片,再以UV光照射固定之。利用Potyvirus、Tobamovirus和Cucumovirus三屬病毒的簡併性引子對,配合actin引子對,以植物樣品全RNA進行multiplex RT-PCR。所增幅並已DIG標定的核酸產物,作為標的物,然後對製備好的晶片進行雜合反應和結果判讀。目前已針對這三屬病毒設計出十四種探針,且由雜合反應結果可知,針對所測試的植物樣品,不論是單獨感染或是複合感染病毒的樣品,皆可被茄科病毒診斷鑑定晶片正確的鑑定出來。此診斷鑑定晶片具有擴充能力,未來可再增加病毒探針數目及種類,以供不同研究目的使用。
Solanaceous plants include many important vegetables and fruits such as tomato, pepper, potato, etc. There are many kinds of plant viruses which can infect solanaceous plants and mixed infection happens frequently in the field. According to the literatures, more than 20 genera 80 species viruses infect solanaceous plant. In Taiwan, there are over 40 viruses including tobamoviruses, potyviruses and cucumoviruses reported to infect solanaceous plants and cause heavy losses. ELISA and RT-PCR are commonly used methods for plant virus detection. Nevertheless, it will be preferable to have a specific and sensitive method which can detect many viruses simultaneously. Therefore, a solanaceous virus chip based on the principle of DNA microarray was attempted to develop. At first we download viral amino acid sequences from GenBank. After using ClustalX software to align amino acid sequences, we used CODEHOP software to separately design degenerate primers of potyviruses, tobamoviruses and cucumoviruses from aligned amino acid sequences. After that, species-specific oligonucleotide probes were designed from the regions between each degenerate primer pair. DIG-labeled or Cy3-labeled targets were prepared from total RNA of infected tissues by means of multiplex RT-PCR with all sets of degenerate primers. Oligonucleotide probes were immobilized onto nylon membrane or slide chip, hybridized with the targets and then the hybridization signals were detected. The results indicated that solanaceous virus chip could correctly identify potyviruses, tobamoviruses and cucumoviruses in single and mixed infection plants. Up to the present, we have designed 14 virus probes for three genera of plant viruses. This virus chip is expandable to include new probes which are required for particular purpose.
目錄………………………………………i
誌謝………………………………………ii
中文摘要…………………………………1
英文摘要…………………………………2
前言………………………………………3
前人研究…………………………………3
生物晶片…………………………………5
病毒介紹…………………………………6
標定方式…………………………………9
材料方法…………………………………11
實驗植物…………………………………11
病毒材料…………………………………11
收集序列資料並設計簡併性引子對……12
種專一性寡核苷酸探針的設計與製備…13
反轉錄反應………………………………14
聚合酶連鎖反應…………………………14
多對引子反轉錄聚合酶連鎖反應………15
標的物之製備……………………………15
逆墨點雜合反應…………………………16
結果………………………………………20
討論………………………………………26
參考文獻…………………………………30
圖…………………………………………37
王添成. 1996. 茄科蔬菜之抗病育種. 健康清潔植物培育研習會專刊. 30-66 pp. 中華民國植物病理學會出版。
李青芸. 2005. 菸草微綠嵌紋病毒引起之辣椒新病害及其感染性選殖株之建構. 國立台灣大學植物病理與微生物學研究所碩士論文.
柯勇. 1998. 作物病害與防治. 117-118. 藝軒圖書出版社. 臺北市. 550 pp.
徐世典、張東柱、張清安、蔡進來、蔡東纂. 2002. 臺灣植物病害名彙(第4版. 386 pp. 中華民國植物病理學會出版)。
徐悅淳. 2001. 重要potyvirus快速鑑定系統之建立. 國立台灣大學植物病理學研究所碩士論文.
陳秀瓊. 2002. 以聚合酶鏈鎖反應結合基因晶片快速偵測犬病毒性疾病. 國立台灣大學獸醫學研究所碩士論文.
黃偉洲. 2001. 海芋嵌紋病毒對彩色海芋生長之影響及其分子檢測系統之建立. 國立台灣大學植物病理學研究所碩士論文.
葉慈容. 2004. 利用逆墨點雜合法鑑定九種馬鈴薯Y屬病毒. 國立台灣大學植物病理與微生物學研究所碩士論文.
農業統計年報. 2005. 行政院農業委員會網站
(http://www.coa.gov.tw/view.php?catid=11180)
Basso, J., Dallaire, P., Charest, P. J., Devantier, Y., and Laliberte, J. F. 1994. Evidence for an internal ribosome entry site within the 5’ non-translated region of turnip mosaic potyvirus RNA. J. Gen. Virol. 75: 3157-3165.
Bertucci, F., Bernard, K., Loriod, B., Chang, Y.-C., Granjeaud, S., Birnbaum, D., Nguyen, C., Peck, K., and Jordan, B. R. 1999. Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples. Hum. Mol. Genet. 8: 1715-1722.
Boonham, N., Walsh, K., Smith, P., Madagan, K., Graham, I., and Barker, I. 2003. Detection of potato viruses using microarray technology: towards a generic method for plant viral disease diagnosis. J. Virol. Methods 108: 181-187.
Chen, J.-J., Wu, R., Yang, P.-C., Huang, J.-Y., Sher, Y.-P., Han, M.-H., Kao, W.-C., Lee, P.-J., Chiu, T.-F., Chang, F., Chu, Y.-W., Wu, C.-W., and Peck, K. 1998. Profiling expression patterns and isolating differentially expressed genes by cDNA microarray system with colorimetry detection. Genomics 51: 313-324.
Carrington, J. C., Jensen, P. E., and Schaad, M. C. 1998. Genetic evidence for an essential role for potyvirus CI protein in cell-to-cell movement. Plant J. 14: 393-400.
Crowther, D. J. 2002. Applications of microarrays in the pharmaceutical industry. Curr. Opin. Pharmacol. 2: 551-554.
Dallot, S., Quiot-Douine, L., Saenz, P., Cervera, M. T., Garcia, J. A., and Quiot, J. B. 2001. Identification of Plum pox virus determinants implicated in specific interactions with different Prunus spp. Phytopathology 91: 159-164.
Dolja, V. V., Haldeman-Cahill, R., Montgomery, A. E., Vandenbosch, K. A., and Carrington, J. C. 1995. Capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch potyvirus. Virology 206: 1007-1016.
Fellers, J., Wan, J., Hong, Y., Collins, G. B., and Hunt, A. G. 1998. In vitro interactions between a potyvirus-encoded, genome-linked protein and RNA-dependent RNA polymerase. J. Gen. Virol. 79: 2043-2049.
Franke-Whittle, I. H., Klammer, S. H., Mayrhofer, S., and Insam, H. 2005. Comparison of different labeling methods for the production of labeled target DNA for microarray hybridization. J. Microbiol. Methods. 65: 117-126.
Fauquet, C. M.., Mayo, M. A.., Maniloff, J., Desselberger, U., Ball, L. A. 2005. Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses. Elsevier Inc., San Diago, USA. 1009-1013; 1053-1054 pp.
Gabig, M., and Wegrzyn, G. 2001. An introduction to DNA chips: principles, technology, applications and analysis. Acta Biochimica Polonica 48: 615-622.
Green, S. K. 1993. Pepper virus researcher in Taiwan and other Asian country. Council of Agriculture Plant Protection Series No. 1. Proceedings of the Symptom on plant Virus and Virus-like Disease. 213-243.
Hirabayashi, J. 2003. Oligosaccharide microarrays for glycomics. Trends Biotechnol. 21: 141–143.
Hughesl, T. R., Mao, M., Jones, A. R., Burchard, J., Martonl, M. J., Shannon, K. W., Lefkowitz, S. M., Ziman, M., Schelterl, J. M., Meyerl, M. R., Kobayashil, S., Davis, C., Dai, H., He, Y. D., Stephaniants, S. B., Cavet, G., Walkerl, W. L., West, A., Coffeyl, E., Shoemaker, D. D., Stoughton, R., Blanchard, A. P., Friend, S. H., and Linsley, P. S. 2001. Expression profiling using microarrays fabricated by an ink-jet Oligonucleotide synthesizer. Nature biotechnology 19: 342-347.
Hsiao, C. R., Huang, L., Bouchara, J.-P., Barton, R., Li, H. C., Chang, T. C. 2005. Identification of medically important molds by an oligonucleotide array. J. Clin. Micorbiol. 43: 3760-8.
Hsu, Y.-C., Yeh, T.-J., and Chang, Y.-C. 2005. A new combination of RT-PCR and reverse dot blot hybridization for rapid detection and identification of potyviruses. J. Virol. Methods 128: 54-60.
Kurian, K. M., Watson, C. J., and Wyllie, A. H. 1999. DNA chip technology. J. Pathol. 187: 267-271.
Lee, G. P., Min, B. E., Kim, C. S., Choi, S. H., Harn, C. H., Kim, S. U., and Ryu, K. H. 2003. Plant virus cDNA chip hybridization for detection and differentiation of four cucurbit-infecting Tobamoviruses. J. Virol. Methods 110: 19-24.
Lee, S. C., and Chang, Y. C. 2006. Multiplex RT-PCR detection of teo orchid viruses with an internal control of plant nad5 mRNA. Plant Pathol. Bull. 15: 187-196.
Liu, F., Sukhacheva, E., Erokhina, T., Schubert, J., and Liu, F. B. 1999. Detection of potyviral nuclear inclusion b proteins by monoclonal antibodies raised to synthetic peptides. Eur. J. Plant Pathol. 105: 389-395.
Liu, S., He, X., Park, G., Josefsson, C., and Perry, K. L. 2002. A conserved capsid protein surface domain of cucumber mosaic virus is essential for efficient aphid vector transmission. J. Virol. 76: 9756-9762.
Marshall, A., and Hodgson, J. 1998. DNA chips: An array of possibilities. Nature biotechnology 16: 27-31
Maughan, N. J., Lewis, F. A., and Smith, V. 2001. An introduction to arrays. Journal of Pathology 195: 3-6.
Moury, B. 2004. Differential selection of genes of cucumber mosaic virus subgroups. Mol. Biol. Evol. 21: 1602-1611.
Nakashima, H., Sako, N., and Hori, K. 1993. Nucleotide sequences of the helper component-proteinase genes of aphid transmissible and non-transmissible isolates of turnip mosaic virus. Arch. Virol. 131: 17-27.
Ng, J. C. K., S. Liu, and K. L. Perry. 2000. Cucumber mosaic virus mutants with altered physical properties and defective in aphid vector transmission. Virology 276: 395-403.
Palukaitis, P., M. J. Roossinck, R. G. Dietzgen, and R. I. B. Francki. 1992. Cucumber mosaic virus. Adv. Virus Res. 41: 281-348.
Predki, P. F. 2004. Functional protein microarrays: ripe for discovery. Curr. Opin. Chem. Biol. 8: 8–13.
Randles, J. W., Palukaitis, P. and Davies, C. 1981. Natural distribution, reasons to continue to name both types TMGMV spread and variation, in the tobacco mosaic virus infecting Nicotiana glauca in Australia. Annals of Applied Biology 98: 109-119.
Riechmann, J. L., Cervera, M. T., and Garcia, J. A. 1995. Processing of the plum pox virus polyprotein at the P3-6K1 junction is not required for virus viability. J. Gen. Virol. 76: 951-956.
Riechmann, J. L., Lain, S., and Garcia, J. A. 1992. Highlights and prospects of potyvirus molecular biology. J. Gen. Virol. 73: 1-16.
Rojas, M. R., Zerbini, F. M., Allison, R. F., Gilbertson, R. L., and Lucas, W. J. 1997. Capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins. Virology 237: 283-295.
Roossinck, M.J., 2002. Evolutionary history of Cucumber mosaic virus deduced by phylogenetic analyses. J. Virol. 76, 3382–3387.
Rose, T. M., Schultz, E. R., Henikoff, J. G., Pietrokovski, S., McCallum, C. M., and Henikoff, S. 1998. Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res. 26: 1628-1635.
Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., Arnheim, N. 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-1354.
Schena, M., Heller, R. A., Theriault, T. P., Konrad, K., Lachenmeier, E., and Davis, R. W. 1998. Microarrays: biotechnology''s discovery platform for functional genomics. Trends Biotechnol. 16: 301-306.
Shoemaker, D. D., and Linsley, P. S. 2002. Recent developments in DNA microarrays. Curr. Opin. Microbiol. 5: 334–337.
Shukla, D. D., and Ward, C. W. 1998. Amino acid sequence homology of coat protein as a basis for identification and classification of the potyvirus group. J. Gen. Virol. 69: 2703-2710.
Soumounou, Y., and Laliberte, J. F. 1994. Nucleic acid-binding properties of the P1 protein of turnip mosaic potyvirus produced in Escherichia coli. J. General Virol. 75: 2567-2573.
Urcuqui-Inchima, S., Haenni, A.-L., and Bernardi, F. 2001. Potyvirus proteins: a wealth of functions. Virus research 74: 157-175.
Walsh, J. A., and Jenner, C. E. 2002. Turnip mosaic virus and the quest for durable resistance. Mol. Plant Pathol. 3: 289-300.
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