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標題: 利用蛋白質體學研究菸草感染竹嵌紋病毒後具有差異性表現之蛋白
Proteomic Analysis of Differential Protein Expression of Bamboo mosaic virus Infected Nicotiana benthamiana
作者: 吳麗琴
Wu, Li-Chin
關鍵字: Bamboo mosaic virus;竹嵌紋病毒;host factors;proteomic;寄主因子;蛋白質體學
出版社: 生物科技學研究所
引用: 林納生、陳脈紀、江濤與林維治。1979。台灣竹類嵌紋病之初步研究。臺灣省林業試驗所試驗報告 第317號。 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。 林民昆。2004。竹嵌紋病毒三基因區蛋白之互補試驗及病毒載體之應用。國立中興大學生物科技學研究所博士論文。 Ahlquist, P. (2006). Parallels among positive-strand RNA viruses, reverse-transcribing viruses and double-stranded RNA viruses. Nat Rev Microbiol 4(5), 371-82. Ahlquist, P., Noueiry, A. O., Lee, W. M., Kushner, D. B., and Dye, B. T. (2003). Host factors in positive-strand RNA virus genome replication. J Virol 77(15), 8181-6. Ashcroft, A. E. (2003). Protein and peptide identification: the role of mass spectrometry in proteomics. Nat Prod Rep 20(2), 202-15. Azpiroz-Leehan, R., and Feldmann, K. A. (1997). T-DNA insertion mutagenesis in Arabidopsis: going back and forth. Trends Genet 13(4), 152-6. Bachem, C. W., van der Hoeven, R. S., de Bruijn, S. M., Vreugdenhil, D., Zabeau, M., and Visser, R. G. (1996). Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J 9(5), 745-53. Bastin, M., and Hall, T. C. (1976). Interaction of elongation factor 1 with aminoacylated brome mosaic virus and tRNA''s. J Virol 20(1), 117-22. Bauer, A., and Kuster, B. (2003). Affinity purification-mass spectrometry. Powerful tools for the characterization of protein complexes. Eur J Biochem 270(4), 570-8. Birnboim, H. C., and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7(6), 1513-23. Blackwell, J. L., and Brinton, M. A. (1997). Translation elongation factor-1 alpha interacts with the 3'' stem-loop region of West Nile virus genomic RNA. J Virol 71(9), 6433-44. Blumenthal, T. (1979). Qbeta RNA replicase and protein synthesis elongation factors EF-Tu and EF-Ts. Methods Enzymol 60, 628-38. Blumenthal, T., and Carmichael, G. G. (1979). RNA replication: function and structure of Qbeta-replicase. Annu Rev Biochem 48, 525-48. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-54. Brown, D., and Gold, L. (1996). RNA replication by Qbeta replicase: a working model. Proc Natl Acad Sci U S A 93(21), 11558-62. Burch-Smith, T. M., Anderson, J. C., Martin, G. B., and Dinesh-Kumar, S. P. (2004). Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39(5), 734-46. Cai, X. Z., Xu, Q. F., Wang, C. C., and Zheng, Z. (2006). Development of a virus-induced gene-silencing system for functional analysis of the RPS2-dependent resistance signalling pathways in Arabidopsis. Plant Mol Biol 62(1-2), 223-32. Cardenas, M. E., Lim, E., and Heitman, J. (1995). Mutations that perturb cyclophilin A ligand binding pocket confer cyclosporin A resistance in Saccharomyces cerevisiae. J Biol Chem 270(36), 20997-1002. Carpentier, S. C., Witters, E., Laukens, K., Deckers, P., Swennen, R., and Panis, B. (2005). Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics 5(10), 2497-507. Casado-Vela, J., Selles, S., and Martinez, R. B. (2006). Proteomic analysis of tobacco mosaic virus-infected tomato (Lycopersicon esculentum M.) fruits and detection of viral coat protein. Proteomics 6, S196-206. Castro, A. J., Carapito, C., Zorn, N., Magne, C., Leize, E., Van Dorsselaer, A., and Clement, C. (2005). Proteomic analysis of grapevine (Vitis vinifera L.) tissues subjected to herbicide stress. J Exp Bot 56(421), 2783-95. 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 ( Pt 5), 1175-9. Chen-Schmeisser, U., and Garrett, R. A. (1977). A new method for the isolation of a 5 S RNA complex with proteins L5, L18 and L25 from Escherichia coli ribosomes. FEBS Lett 74(2), 287-91. Cheng, J. H., Ding, M. P., Hsu, Y. H., and Tsai, C. H. (2001). The partial purified RNA-dependent RNA polymerases from bamboo mosaic potexvirus and potato virus X infected plants containing the template-dependent activities. Virus Res 80(1-2), 41-52. Ciesiolka, J., Lorenz, S., and Erdmann, V. A. (1992). Structural analysis of three prokaryotic 5S rRNA species and selected 5S rRNA--ribosomal-protein complexes by means of Pb(II)-induced hydrolysis. Eur J Biochem 204(2), 575-81. 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(3), 475-83. Cogoni, C., and Macino, G. (2000). Post-transcriptional gene silencing across kingdoms. Curr Opin Genet Dev 10(6), 638-43. Dagert, M., and Ehrlich, S. D. (1979). Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6(1), 23-8. De Nova-Ocampo, M., Villegas-Sepulveda, N., and del Angel, R. M. (2002). Translation elongation factor-1alpha, La, and PTB interact with the 3'' untranslated region of dengue 4 virus RNA. Virology 295(2), 337-47. Dontsova, O., Tishkov, V., Dokudovskaya, S., Bogdanov, A., Doring, T., Rinke-Appel, J., Thamm, S., Greuer, B., and Brimacombe, R. (1994). Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center. Proc Natl Acad Sci U S A 91(10), 4125-9. Douthwaite, S., Garrett, R. A., Wagner, R., and Feunteun, J. (1979). A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25. Nucleic Acids Res 6(7), 2453-70. Dreher, T. W., and Miller, W. A. (2006). Translational control in positive strand RNA plant viruses. Virology 344(1), 185-97. Duguid, J. R., and Dinauer, M. C. (1990). Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection. Nucleic Acids Res 18(9), 2789-92. Ekkapongpisit, M., Wannatung, T., Susantad, T., Triwitayakorn, K., and Smith, D. R. (2007). cDNA-AFLP analysis of differential gene expression in human hepatoma cells (HepG2) upon dengue virus infection. J Med Virol 79(5), 552-61. Erdmann, V. A., Fahnestock, S., Higo, K., and Nomura, M. (1971). Role of 5S RNA in the functions of 50S ribosomal subunits. Proc Natl Acad Sci U S A 68(12), 2932-6. Everberg, H., Leiding, T., Schioth, A., Tjerneld, F., and Gustavsson, N. (2006). Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis. J Chromatogr A 1122(1-2), 35-46. Ferro, M., Seigneurin-Berny, D., Rolland, N., Chapel, A., Salvi, D., Garin, J., and Joyard, J. (2000). Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins. Electrophoresis 21(16), 3517-26. Fu, D. Q., Zhu, B. Z., Zhu, H. L., Jiang, W. B., and Luo, Y. B. (2005). Virus-induced gene silencing in tomato fruit. Plant J 43(2), 299-308. Garrett, R. A., and Noller, H. F. (1979). Structures of complexes of 5S RNA with ribosomal proteins L5, L18 and L25 from Escherichia coli: identification of kethoxal-reactive sites on the 5S RNA. J Mol Biol 132(4), 637-48. Gorg, A., Obermaier, C., Boguth, G., Csordas, A., Diaz, J. J., and Madjar, J. J. (1997). Very alkaline immobilized pH gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins. Electrophoresis 18(3-4), 328-37. Gould, B., and Kramer, E. M. (2007). Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae). Plant Methods 3, 6. Hammond, S. M., Caudy, A. A., and Hannon, G. J. (2001). Post-transcriptional gene silencing by double-stranded RNA. Nat Rev Genet 2(2), 110-9. Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166(4), 557-80. Hartmann, R. K., Vogel, D. W., Walker, R. T., and Erdmann, V. A. (1988). In vitro incorporation of eubacterial, archaebacterial and eukaryotic 5S rRNAs into large ribosomal subunits of Bacillus stearothermophilus. Nucleic Acids Res 16(8), 3511-24. Henzel, W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C., and Watanabe, C. (1993). Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci U S A 90(11), 5011-5. Hileman, L. C., Drea, S., Martino, G., Litt, A., and Irish, V. F. (2005). Virus-induced gene silencing is an effective tool for assaying gene function in the basal eudicot species Papaver somniferum (opium poppy). Plant J 44(2), 334-41. Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G. and Fraley, R. T. (1985). A simple general methad for transferring genes into plants. Virology 181,109-117. Hoving, S., Gerrits, B., Voshol, H., Muller, D., Roberts, R. C., and van Oostrum, J. (2002). Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients. Proteomics 2(2), 127-34. Huang, Y. L., Han, Y. T., Chang, Y. T., Hsu, Y. H., and Meng, M. (2004). Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus. J Virol 78(3), 1271-80. Huber, L. A., Pfaller, K., and Vietor, I. (2003). Organelle proteomics: implications for subcellular fractionation in proteomics. Circ Res 92(9), 962-8. Jiang, Y., Serviene, E., Gal, J., Panavas, T., and Nagy, P. D. (2006). Identification of essential host factors affecting tombusvirus RNA replication based on the yeast Tet promoters Hughes Collection. J Virol 80(15), 7394-404. Joshi, R.L., Ravel, J.M., Haenni, A.L. (1986). Interaction of turnip yellow mosaic virus Val-RNA with eukaryotic elongation factor EF-1a. Search for a function. EMBO J. 5, 1143–1148. Kaiser, J. (2002). Proteomics. Public-private group maps out initiatives. Science 296(5569), 827. Kaloshian, I. (2007). Virus-induced gene silencing in plant roots. Methods Mol Biol 354, 173-81. Komarova, A. V., Real, E., Borman, A. M., Brocard, M., England, P., Tordo, N., Hershey, J. W., Kean, K. M., and Jacob, Y. (2007). Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis. Nucleic Acids Res 35(5), 1522-32. Krysan, P. J., Young, J. C., and Sussman, M. R. (1999). T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11(12), 2283-90. Kushner, D. B., Lindenbach, B. D., Grdzelishvili, V. Z., Noueiry, A. O., Paul, S. M., and Ahlquist, P. (2003). Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus. Proc Natl Acad Sci U S A 100(26), 15764-9. Kyono, K., Miyashiro, M., and Taguchi, I. (2002). Human eukaryotic initiation factor 4AII associates with hepatitis C virus NS5B protein in vitro. Biochem Biophys Res Commun 292(3), 659-66. Lai, M. M. (1998). Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription. Virology 244(1), 1-12. Leong, W. F., and Chow, V. T. (2006). Transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection. Cell Microbiol 8(4), 565-80. 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., 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., 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. 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, J. W., Ding, M. P., Hsu, Y. H., and Tsai, C. H. (2007). Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of Bamboo mosaic virus. Nucleic Acids Res 35(2), 424-32. Lin, N. S. and Chen C. C. (1991). Association of bamboo mosaic virus (BaMV) and BaMV-specific electron-dense crystalline bodies with chloroplasts. Phytopathology, 81, 1551-1555. Lin, N. S., Chai Y.J., Huang T.Y., Chang T.Y., and Hsu Y.H. (l993). Incidence of bamboo mosaic potexvirus in Taiwan. Plant Dis. 77: 448-450. Lin, N. S., and Hsu, Y. H. (1994). A satellite RNA associated with bamboo mosaic potexvirus. Virology 202(2), 707-14. 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 ( Pt 9), 2513-8. Luche, S., Santoni, V., and Rabilloud, T. (2003). Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. Proteomics 3(3), 249-53. Money, T., Reader, S., Qu, L. J., Dunford, R. P., and Moore, G. (1996). AFLP-based mRNA fingerprinting. Nucleic Acids Res 24(13), 2616-7. Montgomery, S. A., Berglund, P., Beard, C. W., and Johnston, R. E. (2006). Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages. J Virol 80(15), 7729-39. Nagy, P. D., and Pogany, J. (2006). Yeast as a model host to dissect functions of viral and host factors in tombusvirus replication. Virology 344(1), 211-20. O''Farrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250(10), 4007-21. Osman, T. A., and Buck, K. W. (1996). Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase. J Virol 70(9), 6227-34. Panavas, T., and Nagy, P. D. (2003). Yeast as a model host to study replication and recombination of defective interfering RNA of Tomato bushy stunt virus. Virology 314(1), 315-25. Panavas, T., Serviene, E., Brasher, J., and Nagy, P. D. (2005). Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses. Proc Natl Acad Sci U S A 102(20), 7326-31. Perez-Bueno, M. L., Rahoutei, J., Sajnani, C., Garcia-Luque, I., and Baron, M. (2004). Proteomic analysis of the oxygen-evolving complex of photosystem II under biotec stress: Studies on Nicotiana benthamiana infected with tobamoviruses. Proteomics 4(2), 418-25. Ratcliff, F., Martin-Hernandez, A. M., and Baulcombe, D. C. (2001). Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25(2), 237-45. Robertson, D. (2004). VIGS vectors for gene silencing: many targets, many tools. Annu Rev Plant Biol 55, 495-519. Rould, M. A., Perona, J. J., and Steitz, T. A. (1991). Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase. Nature 352(6332), 213-8. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular cloning: a laboratory manual, second edition. Cold Spring Harbor Laboratory Press, New York. Sergiev, P., Dokudovskaya, S., Romanova, E., Topin, A., Bogdanov, A., Brimacombe, R., and Dontsova, O. (1998). The environment of 5S rRNA in the ribosome: cross-links to the GTPase-associated area of 23S rRNA. Nucleic Acids Res 26(11), 2519-25. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68(5), 850-8. Shirzadegan, M., Christie, P., and Seemann, J. R. (1991). An efficient method for isolation of RNA from tissue cultured plant cells. Nucleic Acids Res 19(21), 6055. Shpanchenko, O. V., Zvereva, M. I., Dontsova, O. A., Nierhaus, K. H., and Bogdanov, A. A. (1996). 5S rRNA sugar-phosphate backbone protection in complexes with specific ribosomal proteins. FEBS Lett 394(1), 71-5. Stoldt, M., Wohnert, J., Gorlach, M., and Brown, L. R. (1998). The NMR structure of Escherichia coli ribosomal protein L25 shows homology to general stress proteins and glutaminyl-tRNA synthetases. EMBO J 17(21), 6377-84. Verwoerd, T. C., Dekker, B. M., and Hoekema, A. (1989). A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17(6), 2362. Wang, X., Ullah, Z., and Grumet, R. (2000). Interaction between zucchini yellow mosaic potyvirus RNA-dependent RNA polymerase and host poly-(A) binding protein. Virology 275(2), 433-43. Weigel, D., Ahn, J. H., Blazquez, M. A., Borevitz, J. O., Christensen, S. K., Fankhauser, C., Ferrandiz, C., Kardailsky, I., Malancharuvil, E. J., Neff, M. M., Nguyen, J. T., Sato, S., Wang, Z. Y., Xia, Y., Dixon, R. A., Harrison, M. J., Lamb, C. J., Yanofsky, M. F., and Chory, J. (2000). Activation tagging in Arabidopsis. Plant Physiol 122(4), 1003-13. Whitham, S. A., and Wang, Y. (2004). Roles for host factors in plant viral pathogenicity. Curr Opin Plant Biol 7(4), 365-71. Wung, C. H., Hsu, Y. H., Liou, D. Y., Huang, W. C., Lin, N. S. & 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 ( Pt 5), 1119-26. Yamaji, Y., Kobayashi, T., Hamada, K., Sakurai, K., Yoshii, A., Suzuki, M., Namba, S., and Hibi, T. (2006). In vivo interaction between Tobacco mosaic virus RNA-dependent RNA polymerase and host translation elongation factor 1A. Virology 347(1), 100-8. Yang, C. C., Liu J. S., Lin C. P. and Lin N. S. (1997). Nucleotide sequence and phylogenetic analysis of bamboo mosaic potexvirus isolate from common bamboo (Bambusa vulgaris McClure). Bot. Bull. Acad. Sin., 38, 77-84. Yeh, C. C., Cheng A. H. and Hwang H. Y. (1993). Indexing of bamboo mosaic virus and propagation of virus-free bamboo. In: Proceedings of the Symposium on Plant Virus and Viruslike Diseases, Plant Protection Series No. 1. pp. 275-282. R. C. Chiu and Y. Yeh, Eds. Council of Agriculture, R.O.C. Zeenko, V. V., Ryabova, L. A., Spirin, A. S., Rothnie, H. M., Hess, D., Browning, K. S., and Hohn, T. (2002). Eukaryotic elongation factor 1A interacts with the upstream pseudoknot domain in the 3'' untranslated region of tobacco mosaic virus RNA. J Virol 76(11), 5678-91.
竹嵌紋病毒(Bamboo mosaic virus, BaMV),為彎曲絲狀外型,單股正極性的RNA病毒。當病毒感染其寄主後,除了病毒本身表現的蛋白質外,也會利用寄主蛋白質來參與病毒的複製,同時寄主也會產生抵抗病毒複製及傳播的蛋白質。本實驗主要是利用蛋白質體學,分析當菸草被竹嵌紋病毒感染後,有哪些寄主蛋白質的表現量會與未接種菸草產生差異性。由於病毒進入寄主細胞後會嵌入特定胞器膜中進行複製,因此我們先分離出菸草細胞之膜層,接著利用二維電泳將健康及被BaMV感染的菸草膜層蛋白分開,分別比較兩者之間蛋白質分佈的差異,發現存在許多表現量不同的蛋白質,再利用基質輔助雷射脫附離子化-飛行時間質譜儀(MALDI-TOF MS)來鑑定這些蛋白質的身分。找到之差異表現蛋白分別有ribosomal protein L25、RubisCO small chain、calmodulin-1及glycoprotein endopeptidase-like protein。其中ribosomal protein L25只在被BaMV感染的膜層蛋白出現,L25和逆境下會產生的CTC蛋白序列具有同源性。為了暸解L25是否會影響BaMV在植物體內的累積,我們利用菸草脆裂病毒(Tobacco rattle virus, TRV)為載體進行此基因的靜默,經由即時定量聚合酶連鎖反應偵測結果顯示,利用此靜默系統確實可以將L25 mRNA含量降到對照組之25至40 %左右。將此L25基因靜默植物接種病毒,在接種七天後,以西方漬染免疫偵測及北方雜配分析偵測病毒鞘蛋白及RNA的累積,由結果發現L25基因靜默確實會降低竹嵌紋病毒在植物中的累積,但若以狐尾草嵌紋病毒(Foxtail mosaic virus, FoMV)接種此L25基因靜默植物,發現對於狐尾草嵌紋病毒在植物中的累積並無顯著影響。綜合上述結果顯示L25蛋白會影響BaMV在植物中的累積能力,但對FoMV則較並不重要。


Bamboo mosaic virus (BaMV), is a single-stranded positive-sense RNA virus with flexuous rod-shaped morphology. A number of studies have been devoted to analyze the replication of plus-stranded RNA viruses; several host proteins are known to be involved in assembling the viral RNA replication complex, activating the complex for RNA synthesis, and other steps. However, the host has the defense system to against viral replication and spreading which are also mediated through proteins. In this study, we have tried to identify the differentially expressed proteins between mock and BaMV inoculated N. benthamiana leaves by proteomics approach. Since positive-sense RNA virus replication is usually associated with rearrangements of membranous organelles, we first isolated the membrane fraction (P30) of the N. benthamiana leaves. Proteins associated with P30 fraction were resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE) and identified by MALDI-TOF MS. Comparison of protein patterns from P30 fractions of mock and BaMV-infected N. benthamiana in 2-D gels revealed several differential expressed proteins. The differential expression of ribosomal protein L25, RubisCO small chain, calmodulin-1 and glycoprotein endopeptidase-like protein. Among these proteins, ribosomal protein L25 was only found in P30 fractions sample from BaMV-infected plants. It had been reported that ribosomal protein L25 is homologous to general stress proteins CTC. We utilized TRV- based virus-induced gene silencing (VIGS) system to generate L25-knockdown plants and to investigate the effect of this protein on BaMV accumulation. Real-time PCR results showed that the levels of the L25 mRNA in the various L25 knock-down plant were reduced to about 25 and 40 % to those of the control plants. Western blots and northern blots were used to analyze the accumulation of viral coat protein and viral RNA at seven day post-inoculation of BaMV virions. Results showed that the accumulation of BaMV coat protein and viral RNA in L25-knockdown plant were reduced. But no interference on the accumulation of FoMV coat protein was observed in all L25-knockdown plants. Together, these data suggest that L25 likely plays an important role in the BaMV accumulation.

Keywords: Bamboo mosaic virus / host factors / proteomic
其他識別: U0005-1607200713585600
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