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研究生:林信摯
研究生(外文):Shinn-zhi Lin
論文名稱:胡瓜嵌紋病毒2b蛋白促進竹嵌紋病毒在寄主中系統性的移動及感染非寄主性植物
論文名稱(外文):The 2b protein of Cucumber Mosaic Virus Facilitates Bamboo Mosaic Virus Systemic Spread in Host Plants and Infection in Non-host Plants
指導教授:徐堯煇
指導教授(外文):Yau-Heiu Hsu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:45
中文關鍵詞:竹嵌紋病毒胡瓜嵌紋病毒2b蛋白
外文關鍵詞:Bamboo Mosaic Virus (BaMV)Cucumber Mosaic Virus (CMV)2b protein
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胡瓜嵌紋病毒 (Cucumber mosaic cucumovirus, CMV) 寄主範圍相當的廣泛,可感染1,000種以上的植物,其基因體為三條正股的RNAs。經由RNAs序列比對,CMV系統(strains)可區分為三個次族群,分別為IA、 IB 和II。CMV RNA2可以轉錄出一條次基因體RNA 4a,並可轉譯出2b蛋白。2b蛋白能促進CMV在植物中長距離的移動及病徵的表現,並且也干擾植物的抗病機制,包括後轉錄基因靜默(post-transcriptional gene silencing, PTGS) 以及水楊酸誘發的病毒防禦(salicylic acid-mediated virus resistance)。CMV的寄主範圍如此廣泛,2b蛋白可能扮演一個重要的角色。本實驗首次成功的利用細菌大量的表現CMV次族群IA (NT9-CMV) 的2b蛋白並且製備血清,所製備的血清不只可專一的與細菌大量表現出來的2b蛋白作用,並且也可在NT9-CMV感染的植物中偵測到2b蛋白的表現。竹嵌紋病毒 (Bamboo mosaic potexvirus, BaMV) 是potexvirus group 的成員之一,其寄主範圍狹窄,主要感染竹類 (Bambusoideae)以及少數單子葉植物和Nicotiana benthamiana。為了要擴展BaMV的寄主範圍,本實驗將CMV-2b基因選殖於BaMV病毒載體中,構築出BaMV-2b。將此BaMV-2b重組株和BaMV野生株分別接種白藜(Chenopodium quinoa),並收集含有病徵的葉片,各別純化出BaMV-2b 和BaMV病毒,以此病毒作為後續實驗的接種原。兩種接種原等量分別接種在BaMV的寄主植物及多種非寄主植物上,結果發現在寄主植物N. benthamiana上,含有可表現2b蛋白的BaMV-2b病毒移動速度較野生株BaMV病毒快;而在非寄主植物上可發現BaMV-2b病毒可以感染許多野生株BaMV所無法感染的植物,其中包括了: N. rustica, N. tabaccum, N. samsun, Solanum tuberosum L.和Cucumis sativus L.。由實驗證據顯示來自CMV-NT9 的2b蛋白可以促進BaMV在寄主植物中系統性的移動,並且使得BaMV能夠感染其他非寄主性的植物。

Abstract
Cucumber mosaic virus (CMV), infecting a very wide range of plant species (>1,000 species) is a positive-sense RNA plant virus with a tripartite genome. Based on RNA sequence homology, CMV has been divided into subgroup IA, IB and II. The CMV-2b protein which is encoded by a 3’-proximal ORF of the RNA 2 first identified in subgroup II CMV-Q and translated from a subgenomic RNA 4a. CMV-2b protein has the function in facilitating long-distance virus movement and symptom expression and has been identified as a viral counterdefence factor that interferes with the establishment of post- transcriptional gene silencing (PTGS) and salicylic acid-mediated virus resistance in plants. The 2b protein may play an important role in a wide host range of CMV. Previous, attempts by other laboratories to express the 2b protein of subgroup IA or IB in E. coli was unsuccessfully. In this work, we have successfully overexpressed the 2b protein of a subgroup IB CMV, NT9 strain, in E.coli and generated a polyclonal antibody against the 2b protein. The 2b antiserum can interact specifically with 2b protein that not only expressed in E.coli but also accumulated in NT9-CMV-infected plants. Bamboo mosaic virus (BaMV), a member of the potexvirus group with a narrow host range, infects primarily members of the Bambusoideae, few other monocotyledonous plants and Nicotiana benthamiana. In order to extend BaMV host range, we constructed BaMV derivative BaMV-2b by cloning the 2b gene of CMV NT9 strain into the BaMV vector. Using chimeric virus BaMV-2b and wild type BaMV as inocula, virus accumulation and systemic spread in the inoculated plants were compared. Our results suggest that the chimeric virus BaMV-2b engineered to express the 2b protein of CMV facilitated BaMV long distance movement in N. benthamiana (host plant) and enhanced infectivity of BaMV in N. rustica, N. tabaccum, N. samsun, Solanum tuberosum L. and Cucumis sativus L. (non-host plants).

Contents
中文摘要.......................................................2
Abstract.......................................................3
Introduction...................................................4
Materials and Methods..........................................7
Construction of plasmid pET-2b................................7
Mini-preparation of plasmid DNA...............................7
Preparation of competent cells................................8
Transformation................................................8
The 2b protein overexpression and purification from E. coli
cells.......................................................9
Preparation of polyclonal antibody against CMV-2b protein.....9
Construction of plasmid pCB-2b...............................10
Large-scale isolation of plasmid DNA.........................11
Purification of BaMV and BaMV-2b.............................12
Inoculation of plants with viral particles...................13
Total protein extraction from plant tissue...................13
Indirect enzyme-linked immunosorbent assay...................13
Western blot analysis........................................14
Preparation and inoculation of protoplasts...................15
Northern blot hybridization..................................15
Results.......................................................17
Expression and purification of recombinant 2b protein.........17
Production of a polyclonal anti-2b serum and detection of the 2b
protein in plants infected with NT9-CMV.....................17
Construction and infectivity assay of pCB-2b..................18
Effects of CMV-2b gene on the long distance movement of
Bamboo mosaic virus in host plant.............................19
Effects of CMV-2b gene on infectivity of Bamboo mosaic virus in
non-host plants...............................................19
Discussion....................................................21
References....................................................26
Figures.......................................................31

Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R., Mallory, A. C., Smith, T. H., and Vance, V. B. 1998. A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. USA 95:13079-13084.
Andrew, P. L., Guo, H. S., Li, W. X., and Ding, S. W. 2000. Suppression of post- transcriptional gene silencing by a plant viral protein localized in the nucleus. EMBO J. 19:1672-80.
Brigneti, G., Voinnet, O., Li, W. X., Ji, L. H., Ding, S. W., and Baulcombe, D. C. 1998. Viral pathogenicity determinants are supressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17:6739-6746.
Chang, B. Y., Lin, N. S., Liou, D. Y., Chen, J. P., Liou, G. G. and Hus, Y. H. 1997. Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus. J. Gen. Virol. 78:1175-1179.
Chisholm, S. T., Mahajan, S. K., Whitham, S. A., Yamamoto, M. L., and Carrington, J. C. 2000. Cloning of the Arabidopsis RTM1 gene, which controls restriction of long distance movement of tobacco etch virus. Proc. Natl. Sci. USA 97:489-494.
Ding, S. W., Anderson, B. J., Haase, H. R., and Symons, R. H. 1994. New overlapping gene encoded by the cucumber mosaic virus genome. Virology 198:593-601.
Ding, S. W., Li, W. X., and Symons, R. H. 1995a. A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement. EMBO J. 14:5762-5772.
Ding, S. W., Rathjen, J. P., Li, R. S., Swanson, R., Healy, H., and Symons, R. H. 1995b. Efficient infection from cDNA clones of cucumber mosaic cucumovirus RNAs in a new plasmid vector. J. Gen. Virol. 76:459-464.
Ding, S. W., Shi, B. J., Li, W. X., and Symons, R. H. 1996. An interspecies hybrid RNA virus is significantly more virulent than either parental virus. Proc. Natl. Acad. Sci. USA 93:7470-7474.
GÖrlich, D., and Mattaj, I. W. 1996. Nucleocytoplasmic transport. Science 271:1513-1518.
Gallitelli, D. 2000. The ecology of Cucumber mosaic virus and sustainable agriculture. Virus Res. 71:9-21.
Guo, H. S., and Ding, S. W. 2002. A viral protein inhibits the long range signaling activity of the gene silencing signal. The EMBO J. 21:398-407.
Ham, B. K., Lee, T. H., You, J. S., Nam, Y. W., Kim, J. K., Paek, K. H. 1999. Isolation of a putative tobacco host factor interacting with cucumber mosaic virus-encoded 2b protein by yeast two-hybrid screening. Mol Cells 9:548-555.
Hsu, Y. H., Lee, Y. S., Liu, J. S. and Lin N. S. 1998. Differential interactions of bamboo mosaic potexvirus satellite RNAs, helper virus, and host plants. MPMI. 11:1207-1213.
Hsu, Y. H., Wu, C. W., Lin, B. Y., Chen, H. Y., Lee, M. F., and Tsai, C. H. 1995. Complete genomic RNA sequences of cucumber mosaic virus strain NT9 from Taiwan. Archives of Virology. 140:1841-1847.
Hood, J., and Silver, P. A. 1999. In or out? Regulating nuclear transport. Curr. Opin. Cell Biol 11:241-247.
Jans, D. A. 1995. The regulation of protein transport to the nucleus by phosphorylation. Biochem. J. 311:705-716.
Jans, D. A., and Hubner, S. 1996. Regulation of protein transport to the nucleus: central role of phosphorylation. Physiol. Rev. 76:651-685.
Ji, L. H., and Ding, S. W. 2001. The suppressor of transgene RNA silencing encoded by cucumber mosaic virus interferes with salicylic acid-mediated virus resistance. MPMI 14:715-724.
Kasschau, K. D., and Carrington, J. C. 1998. A counterdefensive strategy of plant viruses: Suppression of posttranscriptional gene silencing. Cell 95:461-470.
Li, H. W., Lucy, A. P., Guo, H. S., Li, W. X., Ji, L. H., Wong, S. M., and Ding, S. W. 1999. Strong host resistance targeted against a viral suppressor of the plant gene silencing defense mechanism. EMBO J. 18:2683-2691.
Lin, M. T., Kitajima, E. W., Cupertino, F. P., and Costa, C. L. 1977. Partial purification and some properties of bamboo mosaic virus. Phytopathology 67:1439-1443.
Lin, N. S., and Chen, C. C. 1991. Association of bamboo mosaic virus (BoMV) and BoMV-specific electron-dense crystalline bodies with chloroplasts. Phytopathology 81:1551-1555.
Lin, N. S. and Hsu, Y. H. 1994. A satellite RNA associated with bamboo mosaic potexvirus. Virology. 202:707-714.
Lin, N. S., Lee, Y. S., Lin, B. Y., Lee, C. W. and Hsu, Y. H. 1996. The open reading frame of bamboo mosaic potexvirus satellite RNA is not essential for its replication and can be replaced with a bacterial gene. Proc. Natl. Acad. Sci. USA. 93:3138-3142.
Lin, N. S., Lin, B. Y., Lo, N. W., Hu, C. C., Chow, T. Y., and Hsu, Y. H. 1994. Nucleotide sequence of the genomic RNA of bamboo mosaic potexvirus. J. Gen. Virol. 75:2513-2518.
Lin, N. S., Lin, F. Z., Huang, T. Y., and Hsu, Y. H. 1992. Genome properties of bamboo mosaic virus. Phytopathology. 82:731-734.
Liou, D. Y., Hsu, Y. H., Wung, C. H., Wang, W. H., Lin, N. S. and Chang, B. Y. 2000. Functional analyses and identification of two arginine residues essential to the ATP-utilizing activity of the triple gene block protein 1 bamboo mosaic potexvirus. Virology. 277:336-344.
Liu, J. S., Hsu, Y. H., Huang, T. Y. and Lin N. S. 1997. Molecular evolution and phylogeny of satellite RNA associated with bamboo mosaic potexvirus. J. Mol. Evol. 44:207-213.
Lucy, A. P., Guo, H. S., Li, W. X., and Ding, S. W. 2000. Suppression of post- transcriptional gene silencing by a plant viral protein localized in the nucleus. EMBO J. 19:1672-1680.
Marilyn, J. R., Zhang, L., and Hellwald, K. 1999. Rearrangements in the 5’ nontranslated region and phylogentic analyses of cucumber mosaic virus RNA 3 indicate radial evolution of three subgroups. J. Virol. 73:6752-6758.
Mayers, C. N., Peter, P., and John, P. C. 2000. Subcellular distribution analysis of cucumber mosaic virus 2b protein. J. Gen. Virol. 81:219-226.
Marathe, R., Smith, T. H., Anandalakshmi, R., Bowman, L. H., Fagard, M., Mourrain, P., Vaucheret, H., Vance, V. B. 2000. Plant viral suppressors of post-transcriptional silencing do not suppress transcriptional silencing. Plant J. 22:51-59.
Oparka, K. J., and Santa Cruz, S. 2000. The great escape: phloem transport and unloading of macromolecules. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51:323-347.
Palauqui, J. C., Elmayan, T., Pollien, J. M., and Vaucheret, H. 1997. Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J. 16:4738-4745.
Peden, K. W. C., and Symons, R. H. 1973. Cucumber mosaic virus contains a functionally divided genome. Virology 53:487-492.
Palukaitis, P., Roossinck, M. J., Dietzgen, R. G., and Francki, R. I. B. 1992. Cucumber mosaic virus. Adv. Virus Res. 41:281-348.
Roossinck, M. J., Zhang, L., and Hellwald, K. 1999. Rearrangements in the 5’ nontranslated region and phylogenetic analyses of cucumber mosaic virus RNA 3 indicate radial evolution of three subgroups. J. Virol. 73:6752-6758.
Ruiz-Medrano, R., Xoconostle-Cazares, B., and Lucas, W. J. 2001. The phloem as a conduit for inter-organ communication. Curr. Opin. Plant Biol. 4:202-209.
Szilassy, D., Salanki, K., and Balaz, S. E. 1999. Molecular evidence for the existence of two distinct subgroups in cucumber mosaic cucumovirus. Virus Genes. 18:221-227.
Shi, B. J., Ding, S. W., and Symons, R. H. 1997. In vivo expression of an overlapping gene encoded by the cucumoviruses. J. Gen. Virol. 78:237-241.
Tsai, M. S., Hsu, Y. H., and Lin, N. S. 1999. Bamboo mosaic potexvirus satellite RNA (satBaMV RNA)-encoded p20 protein preferentially binds to satBaMV RNA. J. Virol. 73:3032-3039.
Voinnet, O., and Baulcombe, D. C. 1997. Systemic signaling in gene silencing. Nature 389:553.
Voinnet, O., Vain, P., Angell, S., and Baulcombe, D. C. 1998. Systemic spread of sequence-specific transgene RNA degradation is initiated by localized introduction of ectopic promoterless DNA. Cell 95:177-187.
Voinnet, O., Lederer, C., and Baulcombe, D. C. 2000. A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103:157-167.
Whitham, S. A., Yamamoto, M. L., and Carrington, J. C. 1999. Selectable virus and altered susceptibility mutants in Arabidopsis thaliana. Pro. Natl. Acad. Sci. USA 96: 772-777.
Wung, C. H., Hsu, Y. H., Liou, D. Y., Huang, W. C., Lin, N. S. and Chang, B.Y. 1999. Identification of the RNA-binding sites of the triple gene block protein 1 of bamboo mosaic potexvirus. J. Gen. Virol. 80:1119-1126.
Yang, C. C., Liu, J. S., Lin, C. P. and Lin, N. S. 1997. Nucleotide sequence and phylogenetic analysis of a bamboo mosaic potexvirus isolate from common bamboo (Banbusa vulgaris McClure). Bot. Bull. Acad. Sin. 38:77-84.

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