Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36192
標題: 探討阿拉伯芥疑似甲基轉移酶AT4g18030對竹嵌紋病毒複製的影響以及利用農桿菌滲入法在菸草中表達竹嵌紋病毒蛋白質
The effect of a putative methyltransferase from Arabidopsis thaliana (AT4g18030) on BaMV replication & Expression of BaMV proteins in Nicotiana benthamiana by agroinfiltration
作者: 丁偉哲
Ding, Wei-Jhe
關鍵字: Bamboo Mosaic Virus
農桿菌滲入法
agroinfiltration
methyltransferase
甲基轉移酶
出版社: 生物科技學研究所
引用: 洪有甫。2006。竹嵌紋病毒端帽酵素之純化效率與活性的增進。國立中興大學生物科技學研究所碩士論文。 Anandalakshmi, R., G. J. Pruss, X. Ge, R. Marathe, A. C. Mallory, T. H. Smith, and V. B. Vance. 1998. A viral suppressor of gene silencing in plants. Proc Natl Acad Sci U S A 95:13079-84. Beck, D. L., P. J. Guilford, D. M. Voot, M. T. Andersen, and R. L. Forster. 1991. Triple gene block proteins of white clover mosaic potexvirus are required for transport. Virology 183:695-702. Chang, B. Y., N. S. Lin, D. Y. Liou, J. P. Chen, G. G. Liou, and Y. H. Hsu. 1997. Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus. J Gen Virol 78 :1175-9. Fedorkin, O., A. Solovyev, N. Yelina, A. Zamyatnin, Jr., R. Zinovkin, K. Makinen, J. Schiemann, and S. Yu Morozov. 2001. Cell-to-cell movement of potato virus X involves distinct functions of the coat protein. J Gen Virol 82:449-58. Huang, Y. L., Y. T. Han, Y. T. Chang, Y. H. Hsu, and M. Meng. 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 78:1271-80. Lakatos, L., T. Csorba, V. Pantaleo, E. J. Chapman, J. C. Carrington, Y. P. Liu, V. V. Dolja, L. F. Calvino, J. J. Lopez-Moya, and J. Burgyan. 2006. Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. Embo J 25:2768-80. Li, Y. I., Y. M. Cheng, Y. L. Huang, C. H. Tsai, Y. H. Hsu, and M. Meng. 1998. Identification and characterization of the Escherichia coli-expressed RNA-dependent RNA polymerase of bamboo mosaic virus. J Virol 72:10093-9. Lin, N. S., B. Y. Lin, N. W. Lo, C. C. Hu, T. Y. Chow, and Y. H. Hsu. 1994. Nucleotide sequence of the genomic RNA of bamboo mosaic potexvirus. J Gen Virol 75:2513-8. Marillonnet, S., A. Giritch, M. Gils, R. Kandzia, V. Klimyuk, and Y. Gleba. 2004. In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc Natl Acad Sci U S A 101:6852-7. Outchkourov, N. S., J. Peters, J. de Jong, W. Rademakers, and M. A. Jongsma. 2003. The promoter-terminator of chrysanthemum rbcS1 directs very high expression levels in plants. Planta 216:1003-12. Shiboleth, Y. M., E. Haronsky, D. Leibman, T. Arazi, M. Wassenegger, S. A. Whitham, V. Gaba, and A. Gal-On. 2007. The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development. J Virol 81:13135-48. Wung, C. H., Y. H. Hsu, D. Y. Liou, W. C. Huang, N. S. Lin, and B. Y. Chang. 1999. Identification of the RNA-binding sites of the triple gene block protein 1 of bamboo mosaic potexvirus. J Gen Virol 80 :1119-26. Yang, C. C., J. S. Liu, C. P. Lin, and N. S Lin. 1997. Nucleotide sequence and phylogenetic analysis of a bamboo mosaic potexvirus isolate from common bamboo (Bambusa vulgaris McCure). Bot. Bull. Acad. Sin. 38: 77-84. Yi, G., K. Gopinath, and C. C. Kao. 2007. Selective repression of translation by the brome mosaic virus 1a RNA replication protein. J Virol 81:1601-9. Yoo, S. D., Y. H. Cho, and J. Sheen. 2007. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565-72.
摘要: 竹嵌紋病毒 (Bamboo Mosaic Virus, BaMV) 屬於馬鈴薯病毒群 (Potexvirus group),為單股正意 RNA 病毒;具有 5 個 ORFs (open reading frames),其中的 ORF1 可轉譯出一個分子量約 155 kDa 的複製酵素,由 N 端到 C 端分別為戴帽酵素 (capping enzyme)、 類解旋酶 (helicase-like domain) 和核酸聚合酵素 (RNA-dependent RNA polymerase)。先前的本實驗室已利用酵母雙雜交系統 (yeast two-hybrid system) 找出菸草 (Nicotiana benthamiana) 與竹嵌紋病毒的核酸聚合酵素之間具有交互作用的蛋白質,得到了一個甲基轉移酶 NbMts。目前已有二項證據可以證明 NbMts 會抑制病毒的複製。為了更進一步了解探討,NbMts 對於病毒在宿主中感染複製的響影;我們利用 BLAST 找出阿拉伯芥中與 NbMts 胺基酸序列相似度最高的蛋白質 AT4g18030,以阿拉伯芥的基因 At4g18030 剔除株 (knock-out line) 和野生株 (wide-type) 作為對照,以竹嵌紋病毒感染且分析兩者之間的感染複製差異。目前發現 AT4g18030 似乎對於竹嵌紋病毒在阿拉伯芥中的複製沒有明顯的影響。 另一方面,我們一直期待大量表達與竹嵌紋病毒複製有關的蛋白質以進行相關的研究分析。先前研究就已嘗試利用大腸桿菌、酵母菌和昆蟲細胞三種不同系統來表達竹嵌紋病毒的戴帽酵素、類解旋酶以及核酸聚合酵素,但這些系統都有其不足的地方。我們嘗試以菸草作為宿主以農桿菌滲入法 (agroinfiltration) 的方式感染表達蛋白。就目前的結果看來戴帽酵素有明顯的表達具活性,而類解旋酶和核酸聚合酵素似乎不如預期有所表達。
Bamboo mosaic virus (BaMV) is a positive sense RNA virus of potexvirus group. The 155-kDa viral replicase, encoded by ORF1, comprises an RNA capping enzyme, a helicase-like domain, and an RNA-dependent RNA polymerase (RdRp) domain from N to C termini. According to the result of previous yeast two-hybrid screening, there is an interaction between a putative methyltransfersase (NbMts) of Nicotiana benthamiana and the RdRp of BaMV replicase. The evidence indicate that NbMts inhibit the virus replication in N. benthamiana. An established knockout line of Arabidopsis at At4g18030, presumably a homologue of NbMts, was purchased in this study to investigate the effect of the NbMts on BaMV replication. The degrees of BaMV replication in protoplasts of wild-type or knockout Arabidopsis were compared in this study. The preliminary results showed AT4g18030 didn't affect virus replication in Arabidopsis. In the other aspect, we have desired an over-expression system for a long time for the production of BaMV replicase in order to study its biochemical properties. Systems working in Escherichia coli, Pichia pastori, Sacchacomyes cerevisiae, and insects cells had been tried, but they were far from satisfaction. In this study, we try to express the viral proteins in N. benthamiana by agroinfiltration. To date, we could obtain the active capping enzyme of BaMV by this system, but still failed to obtain the helicase-like domain and the RdRp domain.
URI: http://hdl.handle.net/11455/36192
其他識別: U0005-2907200820570700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2907200820570700
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