Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36230
標題: 建立竹嵌紋病毒移動蛋白二之Nicotiana benthamiana轉基因植物及探討病毒移動蛋白間之交互作用
Generation of Bamboo mosaic virus Triple-gene-block Protein 2 Transgenic Nicotiana benthamiana for Study of Interaction between Viral Movement Proteins
作者: 洪喬
Hung, Ciao
關鍵字: Bamboo mosaic virus;竹嵌紋病毒;movement protein;transgenic N. benthamiana;移動蛋白;基因轉殖植物
出版社: 生物科技學研究所
引用: 余思賢。2007。應用竹嵌紋病毒載體產製雞華氏囊病毒疫苗之研究。國立中興大學生物科技學研究所碩士論文。 林納生、陳脈紀、江濤與林維治。1979。台灣竹嵌紋病毒之初步研究。台灣省林業試驗所試驗報告 第317 號,p. 1-10。 林銘裕。2004。利用竹嵌紋病毒載體表現口蹄疫病毒抗原決定基。國立中興大學生物科技研究所碩士論文。 陳宗賢。2003。應用竹嵌紋病毒載體產製日本腦炎疫苗之研究。國立中興大學生物科技學研究所碩士論文。 廖家德。2000。竹嵌紋病毒病徵表現之遺傳分析及載體構築。國立中興大學農業生物科技學研究所博士論文 蔡佳芬。2003。利用 T-DNA 插入突變法建立 Nicotiana benthamiana 突變株以探討影響竹嵌紋病毒複製之寄主因子。國立中興大學生物科技學研究所碩士論文。 Alzhanova, D. V., Napuli, A. J., Creamer, R. & Dolja, V. V. (2001). Cell-to-cell movement and assembly of a plant closterovirus: roles for the capsid proteins and Hsp70 homolog. EMBO J 20, 6997-7007. Angell, S. M., Davies, C. & Baulcombe, D. C. (1996). Cell-to-cell movement of potato virus X is associated with a change in the size-exclusion limit of plasmodesmata in trichome cells of Nicotiana clevelandii. Virology 216, 197-201. Atabekov, J. G. & Taliansky, M. E. (1990). Expression of a plant virus-coded transport function by different viral genomes. Adv Virus Res 38, 201-248. Azhakanandam, K., Weissinger, S. M., Nicholson, J. S., Qu, R. and Weissinger, A. K. (2007).Amplicon-plus targeting technology (APTT) for rapid production of a highly unstable vaccine protein in tobacco plants. Plant Mol Biol. 63:393-404. Bouzoubaa, S., Ziegler, V., Beck, D., Guilley, H., Richards, K. & Jonard,.G. (1986). Nucleotide sequence of beet necrotic yellow vein virus RNA-2. J Gen Virol 67, 1689-1700. Carrington, J. C.; Kasschau, K. D.; Mahajan, S. K.; Schaad, M. C. Cell-to-Cell and Long-Distance Transport of Viruses in Plants. Plant Cell 8, 1669-1681. Citovsky, V., Knorr, D., Schuster, G. & Zambryski, P. (1990). The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein. Cell 60, 637-647. Citovsky, V., Wong, M. L., Shaw, A. L., Prasad, B. V. & Zambryski, P. (1992). Visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids. Plant Cell 4, 397-411. Cowan, G. H., Lioliopoulou, F., Ziegler, A. & Torrance, L. (2002). Subcellular localisation, protein interactions, and RNA binding of Potato mop-top virus triple gene block proteins. Virology 298, 106-115. Cruz, S. S., Chapman, S., Roberts, A. G., Roberts, I. M., Prior, D. A. & Oparka, K. J. (1996). Assembly and movement of a plant virus carrying a green fluorescent protein overcoat. Proc Natl Acad Sci U S A 93, 6286-6290. Chang, B. Y., Lin, N. S., Liou, D. Y., Chen, J. P., Liou, G. G. & Hsu, Y. H. 1997. Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus. J. Gen. Virol. 78:1175-9. Davies, C., Hills, G. & Baulcombe, D. C. (1993). Sub-cellular localization of the 25-kDa protein encoded in the triple gene block of potato virus X. Virology 197, 166-175. Deom, C. M., Oliver, M. J. & Beachy, R. N. (1987). The 30-Kilodalton Gene Product of Tobacco Mosaic Virus Potentiates Virus Movement. Science 237, 389-394. Erhardt, M., Herzog, E., Lauber, E., Fritsch, C., Guilley, H., Jonard, G., Richards, K. & Bouzoubaa, S. (1999). Transgenic plants expressing the TGB1 protein of peanut clump virus complement movement of TGB1-defective peanut clump virus but not of TGB1-defective beet necrotic yellow vein virus. Plant Cell Rep 18, 614–619. Fedorkin, O., Solovyev, A., Yelina, N., Zamyatnin, A., Jr., Zinovkin, R., Makinen, K., Schiemann, J. & Yu Morozov, S. (2001). Cell-to-cell movement of potato virus X involves distinct functions of the coat protein. J Gen Virol 82, 449-458. Forster, R. L., Beck, D. L., Guilford, P. J., Voot, D. M., Van Dolleweerd, C. J. & Andersen, M. T. (1992). The coat protein of white clover mosaic potexvirus has a role in facilitating cell-to-cell transport in plants. Virology 191, 480-484. Forster, R. L., Bevan, M. W., Harbison, S. A. & Gardner, R. C. (1988). The complete nucleotide sequence of the potexvirus white clover mosaic virus. Nucleic Acids Res 16, 291-303. Forster, R. L., Bevan, M. W., Harbison, S. A. & Gardner, R. C. (1988). The complete nucleotide sequence of the potexvirus white clover mosaic virus. Nucleic Acids Res 16, 291-303. Fujita, M., Mise, K., Kajiura, Y., Dohi, K. & Furusawa, I. (1998). Nucleic acid-binding properties and subcellular localization of the 3a protein of brome mosaic bromovirus. J Gen Virol 79 ( Pt 5), 1273-1280. Fujiwara, T., Giesman-Cookmeyer, D., Ding, B., Lommel, S. A. & Lucas, W. J. (1993). Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein. Plant Cell 5, 1783-1794. Giesman-Cookmeyer, D. & Lommel, S. A. (1993). Alanine scanning mutagenesis of a plant virus movement protein identifies three functional domains. Plant Cell 5, 973-982. Gilbertson, R. L., Sudarshana, M., Jiang, H., Rojas, M. R. & Lucas, W. J. (2003). Limitations on geminivirus genome size imposed by plasmodesmata and virus-encoded movement protein: insights into DNA trafficking. Plant Cell 15, 2578-2591. Hehnle, S., Wege, C. & Jeske, H. (2004). Interaction of DNA with the movement proteins of geminiviruses revisited. J Virol 78, 7698-7706. Herzog, E., Hemmer, O., Hauser, S., Meyer, G., Bouzoubaa, S. & Fritsch, C. (1998). Identification of genes involved in replication and movement of peanut clump virus. Virology 248, 312-322. Hsu, H. T., Chou, Y. L., Tseng, Y. H., Lin, Y. H., Lin, T. M., Lin, N. S., Hsu, Y. H. & Chang, B. Y. (2008). Topological properties of the triple gene block protein 2 of Bamboo mosaic virus. Virology 379, 1-9. Hsu, H. T., Hsu, Y. H., Bi, I. P., Lin, N. S. & Chang, B. Y. (2004). Biological functions of the cytoplasmic TGBp1 inclusions of bamboo mosaic potexvirus. Arch Virol 149, 1027-1035. Hsu, H. T., Tseng, Y. H., Chou, Y. L., Su, S. H., Hsu, Y. H. & Chang, B. Y. (2009). Characterization of the RNA-binding properties of the triple-gene-block protein 2 of Bamboo mosaic virus. Virol J 6, 50. Huisman, M. J., Linthorst, H. J., Bol, J. F. & Cornelissen, J. C. (1988). The complete nucleotide sequence of potato virus X and its homologies at the amino acid level with various plus-stranded RNA viruses. J Gen Virol 69 ( Pt 8), 1789-1798. Hull, R. (1989). The movement of viruses in plant. Annu Rev Phytopathol 27, 213–240. Ivanov, K. I., Ivanov, P. A., Timofeeva, E. K., Dorokhov, Y. L. & Atabekov, J. G. (1994). The immobilized movement proteins of two tobamoviruses form stable ribonucleoprotein complexes with full-length viral genomic RNA. FEBS Lett 346, 217-220. Ju, H. J., Brown, J. E., Ye, C. M. & Verchot-Lubicz, J. (2007). Mutations in the central domain of potato virus X TGBp2 eliminate granular vesicles and virus cell-to-cell trafficking. J Virol 81, 1899-1911. Ju, H. J., Samuels, T. D., Wang, Y. S., Blancaflor, E., Payton, M., Mitra, R., Krishnamurthy, K., Nelson, R. S. & Verchot-Lubicz, J. (2005). The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection. Plant Physiol 138, 1877-1895. Ju, H. J., Ye, C. M. & Verchot-Lubicz, J. (2008). Mutational analysis of PVX TGBp3 links subcellular accumulation and protein turnover. Virology 375, 103-117. Kalinina, N. O., Fedorkin, O. N., Samuilova, O. V., Maiss, E., Korpela, T., Morozov, S. & Atabekov, J. G. (1996). Expression and biochemical analyses of the recombinant potato virus X 25K movement protein. FEBS Lett 397, 75-78. Kalinina, N. O., Rakitina, D. V., Solovyev, A. G., Schiemann, J. & Morozov, S. Y. (2002). RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block. Virology 296, 321-329. Kiselyova, O. I., Yaminsky, I. V., Karger, E. M., Frolova, O. Y., Dorokhov, Y. L. & Atabekov, J. G. (2001). Visualization by atomic force microscopy of tobacco mosaic virus movement protein-RNA complexes formed in vitro. J Gen Virol 82, 1503-1508. Kitajima. E. W., M. T. Lin., F. P. Cupertino and C. L. COSTA. (1975) A mosaic in bamboo of probable viral etiology. Fitopatalogia(Lima, Peru) 10:57 Kobayashi, K., Cabral, S., Calamante, G., Maldonado, S. & Mentaberry, A. (2001). Transgenic tobacco plants expressing the potato virus X open reading frame 3 gene develop specific resistance and necrotic ring symptoms after infection with the homologous virus. Mol Plant Microbe Interact 14, 1274-1285. Krishnamurthy, K., Heppler, M., Mitra, R., Blancaflor, E., Payton, M., Nelson, R. S. & Verchot-Lubicz, J. (2003). The Potato virus X TGBp3 protein associates with the ER network for virus cell-to-cell movement. Virology 309, 135-151. Krishnamurthy, K., Mitra, R., Payton, M. E. & Verchot-Lubicz, J. (2002). Cell-to-cell movement of the PVX 12K, 8K, or coat proteins may depend on the host, leaf developmental stage, and the PVX 25K protein. Virology 300, 269–281. Leonard, D. A. & Zaitlin, M. (1982). A temperature-sensitive strain of tobacco mosaic virus defective in cell-to-cell movement generates an altered viral-coded protein. Virology 117, 416-424. Li, Y. I., Chen, Y. J., Hsu, Y. H. and Meng, M. (2001a). Characterization of the AdoMet-dependent guanylyltransferase activity that is associated with the N terminus of bamboo mosaic virus replicase. J. Virol. 75:782-788. Li, Y. I., Cheng, Y. M., Huang, Y. L., Tsai, C. H., Hsu, Y. H. and Meng, M. (1998). Identification and characterization of the Escherichia coli-expressed RNA-dependent RNA polymerase of bamboo mosaic virus. J. Virol. 72 10093-10099. Li, Y. I., Shih, T. W., Hsu, Y. H., Han, Y. T., Huang, Y. L. and Meng, M. (2001b). The helicase-like domain of plant potexvirus replicase participates in formation of RNA 5’cap structure by exhibiting RNA 5’-triphosphatase activity. J. Virol. 75:12114-12120. Lin, M. K., Chang, B. Y., Liao, J. T., Lin, N. S. & Hsu, Y. H. (2004). Arg-16 and Arg-21 in the N-terminal region of the triple-gene-block protein 1 of Bamboo mosaic virus are essential for virus movement. J Gen Virol 85, 251-259. Lin, M. K., Hu, C. C., Lin, N. S., Chang, B. Y. & Hsu, Y. H. (2006). Movement of potexviruses requires species-specific interactions among the cognate triple gene block proteins, as revealed by a trans-complementation assay based on the bamboo mosaic virus satellite RNA-mediated expression system. J Gen Virol 87, 1357-1367. 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. & 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. U. S. 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. & 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 of bamboo mosaic potexvirus. Virology 277, 336-344. Lough, T. J., Netzler, N. E., Emerson, S. J., Sutherland, P., Carr, F., Beck, D. L., Lucas, W. J. & Forster, R. L. (2000). Cell-to-cell movement of potexviruses: evidence for a ribonucleic protein complex involving the coat protein and first triple gene block protein. Mol Plant–Microbe Interact 13, 962–974. Lough, T. J., Shash, K., Xoconostle-Cazares, B., Hofstra, K. R., Beck, D. L., Balmori, E., Forster, R. L. & Lucas, W. J. (1998).Molecular dissection of the mechanism by which potexvirus triple gene block proteins mediate cell-to-cell transport of infectious RNA.Mol Plant–Microbe Interact 11, 801–814. Lu, H. C., Chen, C. E., Tsai, M. H., Wang, H. I., Su, H. J. & Yeh, H. H. (2009). Cymbidium mosaic potexvirus isolate-dependent host movement systems reveal two movement control determinants and the coat protein is the dominant. Virology 388, 147-159. Malcuit, I., Marano, M. R., Kavanagh, T. A., De Jong, W., Forsyth, A. & Baulcombe, D. C. (1999). The 25-kDa movement protein of PVX elicits Nb-mediated hypersensitive cell death in potato. Mol Plant– Microbe Interact 12, 536–543. Marillonnet, S., Thoeringer, C., Kandzia, R., Klimyuk, V. & Gleba, Y. (2005). Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat Biotechnol 23, 718-723. McGeachy, K. D. & Barker, H. (2000). Potato mop-top virus RNA can move long distance in the absence of coat protein: evidence from resistant, transgenic plants. Mol Plant Microbe Interact 13, 125-128. Mitra, R., Krishnamurthy, K., Blancaflor, E., Payton, M., Nelson, R. S. & Verchot-Lubicz, J. (2003). The potato virus X TGBp2 protein association with the endoplasmic reticulum plays a role in but is not sufficient for viral cell-to-cell movement. Virology 312, 35-48. Morozov, S. Y. & Solovyev, A. G. (2003). Triple gene block: modular design of a multifunctional machine for plant virus movement. J Gen Virol 84, 1351-1366. Morozov, S. Y., Solovyev, A. G., Kalinina, N. O., Fedorkin, O. N., Samuilova, O. V., Schiemann, J. & Atabekov, J. G. (1999). Evidence for two nonoverlapping functional domains in the potato virus X 25K movement protein. Virology 260, 55-63. Morozov, S. Yu., Lukasheva, L. I., Chernov, B. K., Skryabin, K. G. & Atabekov, J. G. (1987). Nucleotide sequence of the open reading frames adjacent to the coat protein cistron in potato virus X genome. FEBS Lett 213, 438–442. Morozov, S., Dolja, V. V. & Atabekov, J. G. (1989). Probable reassortment of genomic elements among elongated RNA-containing plant viruses. J Mol Evol 29, 52-62. Noueiry, A. O., Lucas, W. J. & Gilbertson, R. L. (1994). Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport. Cell 76, 925-932. Ohno, T., Takamatsu, N., Meshi, T., Okada, Y., Nishiguchi, M. & Kiho, Y. (1983). Single amino acid substitution in 30K protein of TMV defective in virus transport function. Virology 131, 255-258. Osman, T. A., Thommes, P. & Buck, K. W. (1993). Localization of a single-stranded RNA-binding domain in the movement protein of red clover necrotic mosaic dianthovirus. J Gen Virol 74 ( Pt 11), 2453-2457. Petty, I. T. & Jackson, A. O. (1990). Mutational analysis of barley stripe mosaic virus RNA beta. Virology 179, 712-718. Rojas, M. R., Noueiry, A. O., Lucas, W. J. & Gilbertson, R. L. (1998). Bean Dwarf mosaic geminivirus movement proteins recognize DNA in a form- and size-specific manner. Cell 95, 105-113. Rupasov, V. V., Morozov, S., Kanyuka, K. V. & Zavriev, S. K. (1989). Partial nucleotide sequence of potato virus M RNA shows similarities to protexviruses in gene arrangement and the encoded amino acid sequences. J Gen Virol 70 ( Pt 7), 1861-1869. Samuels, T. D., Ju, H. J., Ye, C. M., Motes, C. M., Blancaflor, E. B. & Verchot-Lubicz, J. (2007). Subcellular targeting and interactions among the Potato virus X TGB proteins. Virology 367, 375-389. Schepetilnikov, M. V., Manske, U., Solovyev, A. G., Zamyatnin, A. A., Jr., Schiemann, J. & Morozov, S. Y. (2005). The hydrophobic segment of Potato virus X TGBp3 is a major determinant of the protein intracellular trafficking. J Gen Virol 86, 2379-2391. Schmitt, C., Balmori, E., Jonard, G., Richards, K. E. & Guilley, H. (1992). In vitro mutagenesis of biologically active transcripts of beet necrotic yellow vein virus RNA 2: evidence that a domain of the 75-kDa readthrough protein is important for efficient virus assembly. Proc Natl Acad Sci U S A 89, 5715-5719. Sit, T. L. & AbouHaidar, M. G. (1993). Infectious RNA transcripts derived from cloned cDNA of papaya mosaic virus: effect of mutations to the capsid and polymerase proteins. J Gen Virol 74 ( Pt 6), 1133-1140. Skryabin, K. G., Kraev, A. S., Morozov, S., Rozanov, M. N., Chernov, B. K., Lukasheva, L. I. & Atabekov, J. G. (1988). The nucleotide sequence of potato virus X RNA. Nucleic Acids Res 16, 10929-10930. Soellick, T., Uhrig, J. F., Bucher, G. L., Kellmann, J. W. & Schreier, P. H. (2000). The movement protein NSm of tomato spotted wilt tospovirus (TSWV): RNA binding, interaction with the TSWV N protein, and identification of interacting plant proteins. Proc Natl Acad Sci U S A 97, 2373-2378. Solovyev, A. G., Savenkov, E. I., Agranovsky, A. A. & Morozov, S. Y. (1996). Comparisons of the genomic cis-elements and coding regions in RNA beta components of the hordeiviruses barley stripe mosaic virus, lychnis ringspot virus, and poa semilatent virus. Virology 219, 9-18. Taghizadeh, R. R. & Sherley, J. L. (2008). CFP and YFP, but not GFP, provide stable fluorescent marking of rat hepatic adult stem cells. J Biomed Biotechnol 2008, 453590. Tamai, A. & Meshi, T. (2001). Cell-to-cell movement of Potato virus X: the role of p12 and p8 encoded by the second and third open reading frames of the triple gene block. Mol Plant Microbe Interact 14, 1158-1167 Tsai, M. S., Hsu, Y. H., and Lin, N. S. (1999). Bamboo mosaic virus satellite RNA (satBaMV)-encoded protein preferentially binds to satBaMV RNA. J. Virol. 73:2703-2709. Vijaya Palani, P., Kasiviswanathan, V., Chen, J. C., Chen, W., Hsu, Y. H. & Lin, N. S. (2006). The arginine-rich motif of Bamboo mosaic virus satellite RNA-encoded P20 mediates self-interaction, intracellular targeting, and cell-to-cell movement. Mol Plant Microbe Interact 19, 758-767. Voinnet, O., Lederer, C. & Baulcombe, D. C. (2000). A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103, 157-167. Wong, S. M., Lee, K. C., Yu, H. H. & Leong, W. F. (1998). Phylogenetic analysis of triple gene block viruses based on the TGB 1 homolog gene indicates a convergent evolution. Virus Genes 16, 295-302. 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. Yang, Y., Ding, B., Baulcombe, D. C. & Verchot, J. (2000). Cell-to-cell movement of the 25K protein of potato virus X is regulated by three other viral proteins. Mol Plant Microbe Interact 13, 599-605. Yeh, C. C., Chenng, A. H. and Hwang, H. Y. (1993). Indexing of bamboo mosaic virus and propagation of virus-free bamboo plants. Council of Agriculture, Plant Protection Series No.1. Proceedings of the Symposium on Plant Virus and Virus-like Diseases. 275-281.
摘要: 
植物病毒在細胞間的移動需要植物病毒表現的移動蛋白參與,在馬鈴薯病毒屬中,病毒藉由基因體上重疊性的轉譯架構所轉譯出的三個移動蛋白(Triple-gene-block proteins, TGBps1-3)來進行細胞間的移動。屬於馬鈴薯病毒屬之竹嵌紋病毒(Bamboo mosaic virus,BaMV)的TGBp2由於偵測不易,使BaMV移動機制的研究受阻,因此我們使用衛星核酸表現載體系統,將c-myc 胜月太融合TGBp2 N端後,置換部分衛星核酸的轉譯區域,利用農桿菌轉殖技術構築菸草(Nicotiana benthamiana)的TGBp2轉殖植物- sat CP2,以作為大量表現BaMV移動蛋白的載體。經由抗生素與聚合酵素連鎖反應(Polymerase Chain Reaction, PCR)篩選出7株同質體。轉型植物之表現型比較上,除部份轉形株會有葉形較尖等情形,其餘皆與非轉型植物無異。利用北方雜點分析法以及西方墨漬法分析接種BaMV後的轉基因植物,其chimeric satBaMV以及蛋白之表現皆有增加之趨勢。Potexvirus viral RNA在細胞內會和TGBp1及鞘蛋白形成複合體,前往細胞原生質絲(plasmodesmata, PD)並移動至鄰近細胞,但同屬移動蛋白的TGBp2和TGBp3與移動複合體之間的交互作用以及移動機制至今仍未釐清,本實驗利用免疫共沉澱以及酵母菌雙雜交篩選探討鞘蛋白、TGBp1,TGBp2和TGBp3之間的交互作用。免疫共沉澱之結果顯示TGBp1以及TGBp2皆會與BaMV的鞘蛋白有交互作用,但TGBp1與TGBp2無此現象。酵母菌雙雜交篩選的結果顯示CP會與TGBp1或是自身有交互作用。未來可利用 sat CP2轉殖株接種BaMV再藉由免疫螢光染色法來分析c-myc-TGBp2 於細胞內之分佈情形,可更進一步探討BaMV中TGBp2在移動機制中所扮演的角色。

Viral cell-to-cell movement in the host requires assistance of viral-encoded movement protein. Potexviruses contain three overlapping open reading frames(ORF) termed triple gene blocks (TGBs), which encode three movement proteins, designated TGBp1-TGBp3. At present, subcellular localization and the functions of TGBp2 and TGBp3 in viral cell-to-cell movement are still elusive. In this study, we used BaMV TGBp2 transgenic plants to study its interactions with other viral movement proteins and coat protein. Agrobaterium-mediated transformation was used to introduce c-myc tagged TGBp2 gene, constructed in satBaMV expression vector, into Nicotiana benthamiana genome. Antibiotics and polymerase chain reaction was employed to screen the homozygous lines. The phenotypes of TGBp2 transgenic lines are similar to the non-transgenic plants. The accumulation of TGBp2 in transgenic lines was enhanced by inoculating with BaMV, and formed the dimmers and trimers. It has been shown that viral RNA, TGBp1 and CP of Potexvirus form a complex, which moves to plasmodesmata, then translocates to neighboring cell by an unknown mechanism. In order to determine the interactions of TGB proteins and movement complex, we perform co-immunoprecipitation with TGBp1, CP and c-myc antibodies. The results show that TGBp1 could interact with CP, and the c-myc-TGBp2 fusion protein could not interact to CP or TGBp1. In the yeast-two hybridize experiments, we also found that CP could interact with TGBp1 ant itself, but couldn't interact with TGBp2 or TGBp3. Interactions of TGBp2 and TGBp3 are not found in our study.
URI: http://hdl.handle.net/11455/36230
其他識別: U0005-3107200917520300
Appears in Collections:生物科技學研究所

Show full item record
 
TAIR Related Article

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.