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標題: The in vitro study of Nicotiana benthamiana carbonic anhydrase involved in the replication of Bamboo mosaic virus
作者: 吳逸凡
Wu, Yi-Fan
關鍵字: 菸草;bamboo mosaic virus;碳酸酐酶;BaMV;Nicotiana benthamiana;carbonic anhydrase
出版社: 生物科技學研究所
引用: Chen Li-Hung, 2008. The possible functions of chloroplast phosphoglycerate kinase in the life cycle of bamboo mosaic virus infection. Graduate Institute of Biotechnology, National Chung Hsing University. Chen, I.H., Chiu, M.H., Cheng, S.F., Hsu, Y.H., Tsai, C.H., 2013. The glutathione transferase of Nicotiana benthamiana NbGSTU4 plays a role in regulating the early replication of Bamboo mosaic virus. New Phytol 199, 749-757. Cheng, C.W., Hsiao, Y.Y., Wu, H.C., Chuang, C.M., Chen, J.S., Tsai, C.H., Hsu, Y.H., Wu, Y.C., Lee, C.C., Meng, M.S.A., 2009. Suppression of Bamboo Mosaic Virus Accumulation by a Putative Methyltransferase in Nicotiana benthamiana. Journal of virology 83, 5796-5805. Cheng, J.H., Ding, M.P., Hsu, Y.H., 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 research 80, 41-52. Cheng, S.F., Huang, Y.P., Wu, Z.R., Hu, C.C., Hsu, Y.H., Tsai, C.H., 2010. Identification of differentially expressed genes induced by Bamboo mosaic virus infection in Nicotiana benthamiana by cDNA-amplified fragment length polymorphism. BMC Plant Biol 10, 286. Cheng, S.F., Tsai, M.S., Huang, C.L., Huang, Y.P., Chen, I.H., Lin, N.S., Hsu, Y.H., Tsai, C.H., Cheng, C.P., 2013. Ser/Thr Kinase-Like Protein of Nicotiana benthamiana Is Involved in the Cell-to-Cell Movement of Bamboo mosaic virus. PloS one 8. Domsic, J.F., Avvaru, B.S., Kim, C.U., Gruner, S.M., Agbandje-McKenna, M., Silverman, D.N., Mckenna, R., 2008. Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II. Journal of Biological Chemistry 283, 30766-30771. HewettEmmett, D., Tashian, R.E., 1996. Functional diversity, conservation, and convergence in the evolution of the alpha-, beta-, and gamma-carbonic anhydrase gene families. Molecular phylogenetics and evolution 5, 50-77. Huang, Y.W., Hu, C.C., Liou, M.R., Chang, B.Y., Tsai, C.H., Meng, M.H., Lin, N.S., Hsu, Y.H., 2012. Hsp90 Interacts Specifically with Viral RNA and Differentially Regulates Replication Initiation of Bamboo mosaic virus and Associated Satellite RNA. PLoS pathogens 8. Innocenti, A., Leewattanapasuk, W., Muhlschlegel, F.A., Mastrolorenzo, A., Supuran, C.T., 2009. Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the pathogenic yeast Candida glabrata with anions. Bioorganic & medicinal chemistry letters 19, 4802-4805. Innocenti, A., Zimmerman, S., Ferry, J.G., Scozzafava, A., Supuran, C.T., 2004. Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions. Bioorganic & medicinal chemistry letters 14, 4563-4567. Isik, S., Kockar, F., Arslan, O., Guler, O.O., Innocenti, A., Supuran, C.T., 2008. Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the yeast Saccharomyces cerevisiae with anions. Bioorganic & medicinal chemistry letters 18, 6327-6331. Khalifah, R.G., 1973. Carbon-Dioxide Hydration Activity of Carbonic-Anhydrase - Paradoxical Consequences of Unusually Rapid Catalysis. Proceedings of the National Academy of Sciences of the United States of America 70, 1986-1989. Li, Y.I., Chen, Y.J., Hsu, Y.H., Meng, M., 2001a. Characterization of the AdoMet-dependent guanylyltransferase activity that is associated with the N terminus of bamboo mosaic virus replicase. Journal of virology 75, 782-788. Li, Y.I., Cheng, Y.M., Huang, Y.L., Tsai, C.H., Hsu, Y.H., Meng, M., 1998. Identification and characterization of the Escherichia coli-expressed RNA-dependent RNA polymerase of bamboo mosaic virus. Journal of virology 72, 10093-10099. Li, Y.I., Shih, T.W., Hsu, Y.H., Han, Y.T., Huang, Y.L., 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. Journal of virology 75, 12114-12120. Lin, J.W., Ding, M.P., Hsu, Y.H., Tsai, C.H., 2007. Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of Bamboo mosaic virus. Nucleic Acids Res 35, 424-432. Lin, J.W., Hsu, Y.H., Tsai, C.H., 2005. Characterization of the infectivity of Bamboo mosaic virus with its correlation to the in vitro replicase activities in Nicotiana benthamiana. Virus research 112, 77-84. 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. Journal of General Virology 85, 251-259. Lin, M.T., Kitajima, E.W., Cupertino, F.P., 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., Lin, B.Y., Lo, N.W., Hu, C.C., Chow, T.Y., Hsu, Y.H., 1994. Nucleotide-Sequence of the Genomic Rna of Bamboo Mosaic Potexvirus. Journal of General Virology 75, 2513-2518. Lin, N.S., Lin, F.Z., Huang, T.Y., Hsu, Y.H., 1992. Genome Properties of Bamboo Mosaic-Virus. Phytopathology 82, 731-734. Neish, A.C., 1939. Studies on chloroplasts: Their chemical composition and the distribution of certain metabolites between the chloroplasts and the remainder of the leaf. The Biochemical journal 33, 300-308. Prasanth, K.R., Huang, Y.W., Liou, M.R., Wang, R.Y.L., Hu, C.C., Tsai, C.H., Meng, M.H., Lin, N.S., Hsu, Y.H., 2011. Glyceraldehyde 3-Phosphate Dehydrogenase Negatively Regulates the Replication of Bamboo Mosaic Virus and Its Associated Satellite RNA. Journal of virology 85, 8829-8840. Provart, N.J., Majeau, N., Coleman, J.R., 1993. Characterization of Pea Chloroplastic Carbonic-Anhydrase - Expression in Escherichia-Coli and Site-Directed Mutagenesis. Plant Mol Biol 22, 937-943. Rowlett, R.S., 2010. Structure and catalytic mechanism of the beta-carbonic anhydrases. Bba-Proteins Proteom 1804, 362-373. Sawaya, M.R., Cannon, G.C., Heinhorst, S., Tanaka, S., Williams, E.B., Yeates, T.O., Kerfeld, C.A., 2006. The structure of beta-carbonic anhydrase from the carboxysomal shell reveals a distinct subclass with one active site for the price of two. Journal of Biological Chemistry 281, 7546-7555. Slaymaker, D.H., Navarre, D.A., Clark, D., del Pozo, O., Martin, G.B., Klessig, D.F., 2002. The tobacco salicylic acid-binding protein 3 (SABP3) is the.chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response. Proceedings of the National Academy of Sciences of the United States of America 99, 11640-11645. So, A.K.C., Espie, G.S., Williams, E.B., Shively, J.M., Heinhorst, S., Cannon, G.C., 2004. A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell. Journal of bacteriology 186, 623-630. Supuran, C.T., 2010. Carbonic anhydrase inhibitors. Bioorganic & medicinal chemistry letters 20, 3467-3474. Tripp, B.C., Smith, K., Ferry, J.G., 2001. Carbonic anhydrase: New insights for an ancient enzyme. Journal of Biological Chemistry 276, 48615-48618. Tsai Jun-Yuh, 2012. Identification of the involvement of carbonic anhydrase of Nicotiana benthamiana in the Bamboo mosaic virus infection cycle. Graduate Institute of Biotechnology, National Chung Hsing University
Our lab is dedicated to studying host reactions against viral invasion, especially focusing on Bamboo mosaic virus (BaMV), which has been characterized as a flexuous rod-shaped, single-stranded positive-sense RNA virus comprising about 6 thousand nucleotides. Previously, we found over 90 differentially expressed genes after the inoculation of BaMV using cDNA-amplified fragment polymorphism (cDNA-AFLP) technique. One of the fragments, ACAC10-1, was found to help the replication of BaMV since reducing the expression of this gene by virus-induced gene silencing has a driving force to decrease the accumulation of BaMV in inoculated leaves and protoplasts of Nicotiana benthamiana. This gene was cloned according to homologous sequence in N. tabacum and identified as a β-carbonic anhydrase localizing in the chloroplast while the translated product of this gene fragment shows 99% percent identity to the known protein in N. tabacum, and designated as N. benthamiana carbonic anhydrase (NbCA). The interrelationship between NbCA and BaMV was not clearly examined, which makes us curious in unveiling it. NbCA was successfully cloned and expressed in E. coli, and was purified from the soluble fraction of the bacteria after sonication using Ni2+ column. The purified NbCA was then used in an in vitro replication assay by adding to the replicase complex to examine if NbCA can enhance the minus-strand RNA synthesis. The results reveal that NbCA both enhance the plus- and minus-strand RNA synthesis at initiation phase, but not at elongation phase. We then constructed and purified a mutant NbCA, NbCA/C215S, to verify that whether the catalytic site on NbCA contributes to the enhancement of viral replication. The result surprisingly indicate that the catalytic site on CA only affect the initiation of plus-strand RNA synthesis, but not minus-strand RNA synthesis.

本實驗室致力於研究病毒與宿主間的交互作用,並以竹嵌紋病毒 (Bamboo mosaic virus , BaMV)為主力。BaMV為一彎曲絲狀,基因組約六千核苷酸之單一正股RNA病毒。在先前實驗中,我們利用cDNA amplified fragment length polymorphism找出了超過90個在BaMV感染下會有差異性表現的基因。我們發現其中一個名為ACAC10-1的片段在由virus induced gene silencing降低在菸草(Nicotiana benthamiana)中同源性序列的表現量後,在葉片或原生質體(protoplast)中BaMV的累積量有顯著性下降,因此推測此基因之產物很可能會幫助BaMV在菸草中複製。根據在大菸草(Nicotiana tabacum)中一個表現在葉綠體的β-碳酸酐酶(β-carbonic anhydrase)的相似序列,我們成功選殖到此基因的全長,此基因之產物有99%和我們比對到的蛋白序列相同,因此我們將其暫名為N. benthamiana carbonic anhydrase (NbCA)。由於NbCA和BaMV之間的交互作用尚不清楚,因此有待進一步研究。我們於是將NbCA選殖到E. coli中表現,並利用Ni管柱從破菌之後的上清液中純化。接著我們在往後的實驗中將此蛋白加入BaMV replicase complex中一起反應,以確認其是否影響到正股或負股RNA的合成。結果顯示,NbCA同時會影響病毒正股及負股的複製,且是影響在複製起始(initiation),而不會影響在延長作用(elongation)。我們於是又建構並純化了對活性位胺基酸有突變的NbCA/C215S來鑑定是否是此活性位幫助病毒複製,結果意外地顯示,NbCA的催化活性只會影響到病毒正股RNA合成的起始作用,而不會影響到負股RNA合成的起始作用
其他識別: U0005-0608201318012900
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