跳到主要內容

臺灣博碩士論文加值系統

(44.200.77.92) 您好!臺灣時間:2024/02/27 06:05
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:蕭永振
研究生(外文):Yung-Jen Hsiao
論文名稱:細胞自噬參與竹嵌紋病毒感染週期之研究
論文名稱(外文):The study of the role of autophagy in the infection cycle of Bamboo mosaic virus
指導教授:蔡慶修蔡慶修引用關係
口試委員:胡仲祺李珮瑜
口試日期:2011-07-28
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:英文
論文頁數:40
中文關鍵詞:竹嵌紋病毒感染週期細胞自噬
外文關鍵詞:Bamboo mosaic virusBaMVinfection cycleautophagy
相關次數:
  • 被引用被引用:0
  • 點閱點閱:207
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
細胞自噬是真核細胞中高度保留的主要自我降解現象。它利用在細胞質中產生一雙層膜構造將欲降解之物質或胞器包裹形成自噬體(autophagosome),再與胞內具分解力之胞器(如:酵母菌及植物中的液胞或動物中的溶小體)結合進行降解,並將物質分解或清除受損胞器以循環再利用。因此該現象被認為在細胞生長、遭遇逆境或病原入侵時扮演重要角色。在植物中,細胞自噬可協助其生長、對抗逆境及衰老的調控,以維持其正常生理功能及生存。在先前研究中已發現細胞自噬現象參與菸草嵌紋病毒(Tobacco mosaic virus, TMV)感染具NN抗性基因之菸草,以限制過敏性反應(hypersensitive reaction, HR)之訊號,達到控制病毒入侵之目的,並被認為在植物的先天性免疫反應中具有重要調控功能。在此研究中,由於竹嵌紋病毒(Bamboo mosaic virus, BaMV)在感染菸草(Nicotiana benthamiana)時僅產生輕微病徵且不引發過敏性反應。因此我們自菸草中找出細胞自噬過程中的調控蛋白ATG8之同源基因NbLC3。發現NbLC3之表現在受BaMV感染之原生質體高於未接種病毒之原生質體,而病毒鞘蛋白之累積量在NbLC3-knockdown植物上低於健康植物。另一方面我們以螢光標定的方式表現T7-YFP-NbLC3來監測竹嵌紋病毒感染期間,細胞自噬的現象。為了要進一步了解BaMV的感染和細胞自噬的關係,我們使用兩種不同的抑制劑以干擾自噬現象及自噬體的形成。並且接種不同病毒,如:胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV)、菸草嵌紋病毒及蕃茄嵌紋病毒 (Tomato mosaic virus, ToMV)。其結果發現,在加入抑制劑後竹嵌紋病毒與胡瓜嵌紋病毒的複制受到影響,而其他兩種病毒的複制對細胞自噬受到抑制並沒有明顯的改變。綜合以上結果,自噬作用可能在竹嵌紋病毒的感染週期中提供了某種幫助並且在感染週期裡扮演重要角色。

Autophagy is a highly conserved major self-degradation process in eukaryotic cell. During autophagy process, a double-membrane vesicle structure, named autophagosome, appears in the cytoplasm to encapsulate proteins or organelles target for degrading or recycling. The targeted proteins or organelles in the autophagosome will be delivered to lytic compartments, such as vacuole in yeast and plant or lysosome in mammal, by fusion for degradation. Therefore, the autophagy process is thought to play an important role in development or pathological situation. In plant, autophagy is active in its development, starvation, environmental stress, and senescence. From previous study, it has been demonstrated that autophagy is involved in the infection of Tobacco mosaic virus (TMV) and restricting the size expansion of hypersensitive response (HR) lesion to contain the virus inside the lesion in NN Nicotiana benthamiana. They assume that autophagy may regulate plant innate immune response. During Bamboo mosaic virus (BaMV) infection in N. benthamiana plant, it only causes a mosaic symptom but not induces a HR. We identified the ATG8 homologous in the N. benthaminana, named NbLC3 which is a regulatory protein in the autophagosome formation. We found that the expression level of NbLC3 was higher in protoplast of N. benthamiana during BaMV infection and the viral accumulation was decreased in NbLC3-knockdown plant. We also traced the autophagy and autophagsomes in plants during BaMV infection by overexpressed T7-YFP-NbLC3. To understand the possible roles of autophagy in BaMV infection cycle, we blocked autophagic process by inhibitors, Wortmanin and 3-methyladenine (3-MA), and infected with different viruses, BaMV, Cucumber Mosaic Virus (CMV), TMV, and Tomato Mosaic Virus (ToMV). Only BaMV and CMV showed susceptible to the interference of autophagy and the others showed that their replication was not affect by inhibitors. Finally, these result suggested that autophagy may play an important role during BaMV infection.

Contents

中文摘要 i
Abstract ii
Introduction 1
Bamboo mosaic virus, BaMV 1
Autopahgy 1
Viral interaction with autophagy 2
Autophagy may participate in the life cycle of plant virus 3
Materials and Methods 5
Identification of NbLC3 gene 5
Plasmid construction 5
Transient expression of NbLC3 6
Immunoprecipitation 6
Protoplast isolation and inoculation 7
Protein extraction and western blot 8
Plant RNA extraction 8
Reverse transcription 9
Real-time PCR 9
Confocal microscopy 10
Results 11
The identification of ATG8 homolog, NbLC3, in Nicotiana benthamiana 11
NbLC3 might be involved in BaMV infection cycle 11
YFP-NbLC3 could successfully localiz to autophagosome in N. benthamiana 12
Autophagy could help the accumulation of BaMV 13
Discussion 15
Conclusion 17
Figures 18
References 33



Bassham, D. C., Laporte, M., Marty, F., Moriyasu, Y., Ohsumi, Y., Olsen, L. J., and Yoshimoto, K. (2006). Autophagy in development and stress responses of plants. Autophagy 2(1), 2-11.
Biederbick, A., Kern, H. F., and Elsasser, H. P. (1995). Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66(1), 3-14.
Blommaart, E. F., Krause, U., Schellens, J. P., Vreeling-Sindelarova, H., and Meijer, A. J. (1997). The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem 243(1-2), 240-6.
Cantley, L. C. (2002). The phosphoinositide 3-kinase pathway. Science 296(5573), 1655-7.
Chung, T., Suttangkakul, A., and Vierstra, R. D. (2009). The ATG autophagic conjugation system in maize: ATG transcripts and abundance of the ATG8-lipid adduct are regulated by development and nutrient availability. Plant Physiol 149(1), 220-34.
Cillo, F., Roberts, I. M., and Palukaitis, P. (2002). In situ localization and tissue distribution of the replication-associated proteins of Cucumber mosaic virus in tobacco and cucumber. J Virol 76(21), 10654-64.
Contento, A. L., Xiong, Y., and Bassham, D. C. (2005). Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein. Plant J 42(4), 598-608.
Cruz, S. S., Roberts, A. G., Prior, D. A., Chapman, S., and Oparka, K. J. (1998). Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions. Plant Cell 10(4), 495-510.
Dreux, M., Gastaminza, P., Wieland, S. F., and Chisari, F. V. (2009). The autophagy machinery is required to initiate hepatitis C virus replication. Proc Natl Acad Sci U S A 106(33), 14046-51.
Drose, S., Bindseil, K. U., Bowman, E. J., Siebers, A., Zeeck, A., and Altendorf, K. (1993). Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases. Biochemistry 32(15), 3902-6.
Geng, J., and Klionsky, D. J. (2008). The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. ''Protein modifications: beyond the usual suspects'' review series. EMBO Rep 9(9), 859-64.
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95-98.
Hanada, T., Satomi, Y., Takao, T., and Ohsumi, Y. (2009). The amino-terminal region of Atg3 is essential for association with phosphatidylethanolamine in Atg8 lipidation. FEBS Lett 583(7), 1078-83.
Hanaoka, H., Noda, T., Shirano, Y., Kato, T., Hayashi, H., Shibata, D., Tabata, S., and Ohsumi, Y. (2002). Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiol 129(3), 1181-93.
Hayward, A. P., and Dinesh-Kumar, S. P. (2010). What can Plant Autophagy Do for an Innate Immune Response? Annu Rev Phytopathol.
Hayward, A. P., Tsao, J., and Dinesh-Kumar, S. P. (2009). Autophagy and plant innate immunity: Defense through degradation. Semin Cell Dev Biol 20(9), 1041-7.
Huang, S. C., Chang, C. L., Wang, P. S., Tsai, Y., and Liu, H. S. (2009). Enterovirus 71-induced autophagy detected in vitro and in vivo promotes viral replication. J Med Virol 81(7), 1241-52.
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.
Huss, M., Ingenhorst, G., Konig, S., Gassel, M., Drose, S., Zeeck, A., Altendorf, K., and Wieczorek, H. (2002). Concanamycin A, the specific inhibitor of V-ATPases, binds to the V(o) subunit c. J Biol Chem 277(43), 40544-8.
Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., and Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19(21), 5720-8.
Klionsky, D. J. (2007). Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 8(11), 931-7.
Klionsky, D. J., Abeliovich, H., Agostinis, P., Agrawal, D. K., Aliev, G., Askew, D. S., Baba, M., Baehrecke, E. H., Bahr, B. A., Ballabio, A., Bamber, B. A., Bassham, D. C., Bergamini, E., Bi, X., Biard-Piechaczyk, M., Blum, J. S., Bredesen, D. E., Brodsky, J. L., Brumell, J. H., Brunk, U. T., Bursch, W., Camougrand, N., Cebollero, E., Cecconi, F., Chen, Y., Chin, L. S., Choi, A., Chu, C. T., Chung, J., Clarke, P. G., Clark, R. S., Clarke, S. G., Clave, C., Cleveland, J. L., Codogno, P., Colombo, M. I., Coto-Montes, A., Cregg, J. M., Cuervo, A. M., Debnath, J., Demarchi, F., Dennis, P. B., Dennis, P. A., Deretic, V., Devenish, R. J., Di Sano, F., Dice, J. F., Difiglia, M., Dinesh-Kumar, S., Distelhorst, C. W., Djavaheri-Mergny, M., Dorsey, F. C., Droge, W., Dron, M., Dunn, W. A., Jr., Duszenko, M., Eissa, N. T., Elazar, Z., Esclatine, A., Eskelinen, E. L., Fesus, L., Finley, K. D., Fuentes, J. M., Fueyo, J., Fujisaki, K., Galliot, B., Gao, F. B., Gewirtz, D. A., Gibson, S. B., Gohla, A., Goldberg, A. L., Gonzalez, R., Gonzalez-Estevez, C., Gorski, S., Gottlieb, R. A., Haussinger, D., He, Y. W., Heidenreich, K., Hill, J. A., Hoyer-Hansen, M., Hu, X., Huang, W. P., Iwasaki, A., Jaattela, M., Jackson, W. T., Jiang, X., Jin, S., Johansen, T., Jung, J. U., Kadowaki, M., Kang, C., Kelekar, A., Kessel, D. H., Kiel, J. A., Kim, H. P., Kimchi, A., Kinsella, T. J., Kiselyov, K., Kitamoto, K., Knecht, E., Komatsu, M., Kominami, E., Kondo, S., Kovacs, A. L., Kroemer, G., Kuan, C. Y., Kumar, R., Kundu, M., Landry, J., Laporte, M., Le, W., Lei, H. Y., Lenardo, M. J., Levine, B., Lieberman, A., Lim, K. L., Lin, F. C., Liou, W., Liu, L. F., Lopez-Berestein, G., Lopez-Otin, C., Lu, B., Macleod, K. F., Malorni, W., Martinet, W., Matsuoka, K., Mautner, J., Meijer, A. J., Melendez, A., Michels, P., Miotto, G., Mistiaen, W. P., Mizushima, N., Mograbi, B., Monastyrska, I., Moore, M. N., Moreira, P. I., Moriyasu, Y., Motyl, T., Munz, C., Murphy, L. O., Naqvi, N. I., Neufeld, T. P., Nishino, I., Nixon, R. A., Noda, T., Nurnberg, B., Ogawa, M., Oleinick, N. L., Olsen, L. J., Ozpolat, B., Paglin, S., Palmer, G. E., Papassideri, I., Parkes, M., Perlmutter, D. H., Perry, G., Piacentini, M., Pinkas-Kramarski, R., Prescott, M., Proikas-Cezanne, T., Raben, N., Rami, A., Reggiori, F., Rohrer, B., Rubinsztein, D. C., Ryan, K. M., Sadoshima, J., Sakagami, H., Sakai, Y., Sandri, M., Sasakawa, C., Sass, M., Schneider, C., Seglen, P. O., Seleverstov, O., Settleman, J., Shacka, J. J., Shapiro, I. M., Sibirny, A., Silva-Zacarin, E. C., Simon, H. U., Simone, C., Simonsen, A., Smith, M. A., Spanel-Borowski, K., Srinivas, V., Steeves, M., Stenmark, H., Stromhaug, P. E., Subauste, C. S., Sugimoto, S., Sulzer, D., Suzuki, T., Swanson, M. S., Tabas, I., Takeshita, F., Talbot, N. J., Talloczy, Z., Tanaka, K., Tanida, I., Taylor, G. S., Taylor, J. P., Terman, A., Tettamanti, G., Thompson, C. B., Thumm, M., Tolkovsky, A. M., Tooze, S. A., Truant, R., Tumanovska, L. V., Uchiyama, Y., Ueno, T., Uzcategui, N. L., van der Klei, I., Vaquero, E. C., Vellai, T., Vogel, M. W., Wang, H. G., Webster, P., Wiley, J. W., Xi, Z., Xiao, G., Yahalom, J., Yang, J. M., Yap, G., Yin, X. M., Yoshimori, T., Yu, L., Yue, Z., Yuzaki, M., Zabirnyk, O., Zheng, X., Zhu, X., and Deter, R. L. (2008). Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4(2), 151-75.
Levine, B., and Deretic, V. (2007). Unveiling the roles of autophagy in innate and adaptive immunity. Nat Rev Immunol 7(10), 767-77.
Levine, B., and Klionsky, D. J. (2004). Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6(4), 463-77.
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(2), 782-8.
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(12), 10093-9.
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(24), 12114-20.
Lin, L. T., Dawson, P. W., and Richardson, C. D. (2010). Viral interactions with macroautophagy: a double-edged sword. Virology 402(1), 1-10.
Lin, M. K., Chang, B. Y., Liao, J. T., Lin, N. S., and 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(Pt 1), 251-9.
Lin, M. K., Hu, C. C., Lin, N. S., Chang, B. Y., and 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. Journal of General Virology 87(Pt 5), 1357-67.
Lin, N. S., Chai, Y. J., Huang, T. Y., Chang, T. Y., and Hsu, Y. H. (1993). Incidence of bamboo mosaic potexvirus in Taiwan. Plant Disease 77(5), 448-450.
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. Journal of General Virology 75 ( Pt 9), 2513-8.
Lin, N. S., Lin, F. Z., Huang, T. Y., and Hsu, Y. H. (1992). Gemone properties of bambbo mosaic virus. Phytopathology 82(7), 731-734.
Liu, Y., Schiff, M., Czymmek, K., Talloczy, Z., Levine, B., and Dinesh-Kumar, S. P. (2005). Autophagy regulates programmed cell death during the plant innate immune response. Cell 121(4), 567-77.
Mas, P., and Beachy, R. N. (1999). Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA. J Cell Biol 147(5), 945-58.
Mijaljica, D., Prescott, M., and Devenish, R. J. (2006). Endoplasmic reticulum and Golgi complex: Contributions to, and turnover by, autophagy. Traffic 7(12), 1590-5.
Miller, S., and Krijnse-Locker, J. (2008). Modification of intracellular membrane structures for virus replication. Nat Rev Microbiol 6(5), 363-74.
Mizui, T., Yamashina, S., Tanida, I., Takei, Y., Ueno, T., Sakamoto, N., Ikejima, K., Kitamura, T., Enomoto, N., Sakai, T., Kominami, E., and Watanabe, S. (2010). Inhibition of hepatitis C virus replication by chloroquine targeting virus-associated autophagy. J Gastroenterol 45(2), 195-203.
Rust, R. C., Landmann, L., Gosert, R., Tang, B. L., Hong, W., Hauri, H. P., Egger, D., and Bienz, K. (2001). Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex. J Virol 75(20), 9808-18.
Seay, M., Patel, S., and Dinesh-Kumar, S. P. (2006). Autophagy and plant innate immunity. Cell Microbiol 8(6), 899-906.
Seglen, P. O., and Gordon, P. B. (1982). 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A 79(6), 1889-92.
Subramaniam, S. (1998). The Biology Workbench--a seamless database and analysis environment for the biologist. Proteins 32(1), 1-2.
Suhy, D. A., Giddings, T. H., Jr., and Kirkegaard, K. (2000). Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles. J Virol 74(19), 8953-65.
Tanida, I., Fukasawa, M., Ueno, T., Kominami, E., Wakita, T., and Hanada, K. (2009). Knockdown of autophagy-related gene decreases the production of infectious hepatitis C virus particles. Autophagy 5(7), 937-45.
Wong, J., Zhang, J., Si, X., Gao, G., Mao, I., McManus, B. M., and Luo, H. (2008). Autophagosome supports coxsackievirus B3 replication in host cells. J Virol 82(18), 9143-53.
Yoshimoto, K. (2004). Processing of ATG8s, Ubiquitin-Like Proteins, and Their Deconjugation by ATG4s Are Essential for Plant Autophagy. The Plant Cell Online 16(11), 2967-2983.


QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
1. 竹嵌紋病毒類解旋酵素之特定胺基酸對酵素活性及病毒於白蔾內存活的影響
2. 菸草呼吸爆發氧化酶B對於竹嵌紋病毒複製之影響
3. 竹嵌紋病毒顆粒與含有三重疊基因區第三轉譯蛋白之病毒移動複合體之穩定結合
4. 竹嵌紋病毒戴帽酵素中特定胺基酸殘基定點突變及特定區域截斷對轉甲基酵素活性及AdoMet依賴性鳥嘌呤核苷基轉移酵素活性之影響
5. 竹嵌紋病毒負股3'端與核酸解螺旋酵素之結合分析
6. 提升竹嵌紋病毒載體平台產製日本腦炎疫苗的效率
7. 菸草去氧-D-木酮醣-5-磷酸還原異構酶參與竹嵌紋病毒複製之研究
8. 追蹤竹嵌紋病毒在細胞內的移動以及自噬作用對竹嵌紋病毒的影響之探討
9. 利用生體外複製系統研究菸草碳酸酐酶與竹嵌紋病毒複製之關係
10. 菸草基因NbRab7參與竹嵌紋病毒感染週期之研究
11. I:人類 6-磷酸葡萄糖異構酵素與鳥苷三磷酸結合及其遺傳性突變之結構與功能關聯性研究 II: 竹嵌紋病毒戴帽酵素中保留性組胺酸於形成[酵素-m7GMP]中間產物時之功能分析
12. I:人類 6-磷酸葡萄糖異構酵素與鳥苷三磷酸結合及其遺傳性突變之結構與功能關聯性研究II: 竹嵌紋病毒戴帽酵素中保留性組胺酸於形成[酵素-m7GMP]中間產物時之功能分析
13. 竹嵌紋病毒三重疊基因區第三轉譯蛋白之表現、純化及特性分析
14. 竹嵌紋病毒缺失性RNA包被訊號之研究