BYL无细胞系统对植物RNA病毒翻译复制机制的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2017-0514

摘要/Abstract

摘要： 无细胞翻译系统是高效表达重组蛋白质的体外分子研究系统，BYL是一种通过密度梯度离心法脱去液泡的烟草BY-2裂解液无细胞系统。BYL系统可支持外源mRNA以及多种植物RNA病毒的蛋白质表达，由于BYL系统中保留植物RNA病毒复制所需的胞内膜结构，所以该系统可支持RNA病毒核酸负义链、正义链和亚基因RNA的合成。综述了BYL系统应用于植物RNA病毒翻译和复制等机理以及RNA干扰机制方面的研究进展，并对其研究前景进行展望，旨在便于更多的研究者系统了解BYL无细胞系统，将其更广泛地应用于其他RNA病毒的翻译复制机制研究中。

关键词: 无细胞体系, BYL, 蛋白质表达, 植物RNA病毒

Abstract: Cell-free translation system is an in vitro molecular study system for efficient expression of recombinant proteins. The BYL is tobacco BY-2 lysate cell-free system from which vacuoles are removed by density gradient centrifugation. The BYL system supports recombinant protein expression and many plant viral protein expressions. Because the intracellular membrane fraction required for viral RNA replication is retained，the BYL system also supports negative-strand，positive-strand and subgenomic RNA synthesis of plant positive-strand RNA viruses. This paper reviewed the applications of BYL system in the molecular insight of translation and replication mechanism of plant RNA viruses as well as RNA interference mechanism，and prospected their future research trend. This review systematically illustrated the BYL system，which is expected to be applied in the study of of other plant RNA viruses and provides intriguing insights.

Key words: cell-free system, BYL, protein expression, plant RNA virus

安梦楠, 吴元华. BYL无细胞系统对植物RNA病毒翻译复制机制的研究进展[J]. 生物技术通报, 2017, 33(12): 45-50.

AN Meng-nan, WU Yuan-hua. Research Progress on BYL Cell-free Systems in Plant RNA Virus Translation and Replication Mechanisms[J]. Biotechnology Bulletin, 2017, 33(12): 45-50.

参考文献

［1］Kwon YC, Jewett MC. High-throughput preparation methods of crude extract for robust cell-free protein synthesis［J］. Sci Rep, 2015, 5：8663.
［2］Swartz JR. Transforming biochemical engineering with cell-free biology［J］. AIChE Journal, 2012, 58（1）：5-13.
［3］Buntru M, Vogel S, Spiegel H, et al. Tobacco BY-2 cell-free lysate：an alternative and highly-productive plant-based in vitro translation system［J］. BMC Biotechnology, 2014, 14（37）：1-17.
［4］Caschera F, Noireaux V. Synthesis of 2. 3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system［J］. Biochimie, 2014, 99：162-168.
［5］Nirenberg MW, Matthael JH. The Dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides［J］. Proceedings of the National Academy of Sciences, 1961, 47：1588-1602.
［6］Carlson ED, Gan R, Hodgman CE, et al. Cell-free protein synthesis：Applications come of age［J］. Biotechnology Advances, 2012, 30（5）：1185-1194.
［7］Hino M, Kataoka M, Kajimoto K, et al. Efficiency of cell-free protein synthesis based on a crude cell extract from Escherichia coli, wheat germ, and rabbit reticulocytes［J］. J Biotechnol, 2008, 133：183-189.
［8］Komoda K, Naito S, Ishikawa M. Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts［J］. Proceedings of the National Academy of Sciences, 2004, 101（7）：1863-1867.
［9］Ishibashi K, Ishikawa M. Replication of tobamovirus RNA［J］. Annual Review of Phytopathology, 2016, 4（54）：55-78.
［10］ Takamatsu N, Watanabe Y, Iwasaki T, et al. Deletion analysis of the 5’ untranslated leader sequence of Tobacco mosaic virus RNA［J］. Journal of Virology, 1991, 65（3）：1619-1622.
［11］Molla A, Paul AV, Wimmer E. Cell-free, de novo synthesis of poliovirus［J］. Science, 1991, 254：1647-1651.
［12］Herold J and Andino R. Poliovirus RNA replication requires genome circularization through a protein-protein bridge［J］. Molecular Cell, 2001, 7：581-591.
［13］Barton DJ, Morasco BJ, Flanegan JB. Translating ribosomes inhibit poliovirus negative-strand RNA synthesis［J］. Journal of Virology, 1999, 73（12）：10104-10112.
［14］Reuter LJ, Bailey MJ, Joensuu JJ, et al. Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells［J］. Plant Biotechnology Journal, 2014, 12（4）：402-410.
［15］Nambo M, Kurihara D, Yamada T, et al. Combination of synthetic chemistry and live-cell imaging identified a rapid cell division inhibitor in tobacco and Arabidopsis thaliana［J］. Plant Cell Physiology, 2016, 57（11）：2255-2268.
［16］Chang JJ, Sonobe S, Shibaoka H. Assembly of microtubules in a cytoplasmic extract of tobacco BY-2 mini protoplasts in the absence of microtubule-stabilizing agents［J］. Plant Cell Physiology, 1992, 33（4）：497-501.
［17］Iwakawa HO, Kaido M, Mise K, et al. cis-Acting core RNA elements required for negative-strand RNA synthesis and cap-independent translation are separated in the 3’-untranslated region of Red clover necrotic mosaic virus RNA1［J］. Virology, 2007, 369：168-181.
［18］An M, Iwakawa HO, Mine A, et al. A Y-shaped RNA structure in the 3’ untranslated region together with the trans-activator and core promoter of Red clover necrotic mosaic virus RNA2 is required for its negative-strand RNA synthesis［J］. Virology, 2010, 405：100-109.
［19］Komoda K, Mawatari N, Hagiwara-Komoda Y, et al. Identification of a ribonucleoprotein intermediate of tomato mosaic virus RNA replication complex formation［J］. Journal of Virology, 2007, 81（6）：2584-2591.
［20］Chen J, Ahlquist P. Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1a［J］. Journal of Virology, 2000, 74：4310-4318.
［21］Ishibashi K, Masuda K, Naito S, et al. An inhibitor of viral RNA replication is encoded by a plant resistance gene［J］. Proceedings of the National Academy of Sciences, 2007, 104：13833-13838.
［22］Okamoto K, Nagano H, Iwakawa, HO, et al. cis-Preferential requirement of a-1 frameshift product p88 for the replication of Red clover necrotic mosaic virus RNA1［J］. Virology, 2008, 375（1）：205-212.
［23］Mizumoto H, Tatsuta M, Kaido M, et al. Cap independent translational enhancement by the 3’ untranslated region of Red clover necrotic mosaic virus RNA1［J］. Journal of Virology, 2003, 77（22）：12113-12121.
［24］Iwakawa HO, Mine A, Hyodo K, et al. Template recognition mechanisms by replicase proteins differ between bipartite positive-strand genomic RNAs of a plant virus［J］. Journal of Virology, 2011, 85（1）：497-509,
［25］Iwakawa HO, Tajima Y, Taniguchi T, et al. Poly（A）-binding protein facilitates translation of an uncapped/ nonpolyadenylated viral RNA by binding to the 3’ untranslated region［J］. Journal of Virology, 2012, 86（15）：7836-7849.
［26］Mine A, Takeda A, Taniguchi T, et al. Identification and characterization of the 480-kilodalton template-specific RNA dependent RNA polymerase complex of Red clover necrotic mosaic virus［J］. Journal of Virology, 2010, 84（12）：6070-6081.
［27］Ishibashi K, Masuda K, Naito S, et al. An inhibitor of viral RNA replication is encoded by a plant resistance gene［J］. Proceedings of the National Academy of Sciences, 2007, 104（34）：13833-13838.
［28］Gursinsky T, Schulz B, Behrens SE. Replication of Tomato bushy stunt virus RNA in a plant in vitro system［J］. Virology, 2009, 390：250-260.
［29］Roberts BE, Paterson BM. Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ［J］. Proceedings of the National Academy of Sciences, 1973, 70（8）：2330-2334.
［30］刘永祥, 陈思远, 张逸婧, 等. 麦胚无细胞蛋白合成系统研究进展［J］. 食品工业科技, 2015, 36（17）：379-383.
［31］Guo L, Allen EM, Miller WA. Base-pairing between untranslated regions facilitates translation of uncapped, nonpolyadenylated viral RNA［J］. Molecular Cell, 2001, 7（5）：1103-1109.
［32］Shen RZ, Rakotondrafara AM, Miller WA. trans Regulation of cap-independent translation by a viral subgenomic RNA［J］. Journal of Virology, 2006, 80（20）：10045-10054.
［33］Treder K, Pettit Kneller EL, Allen EM, et al. The 3’ cap-independent translation element of Barley yellow dwarf virus binds eIF4F via the eIF4G subunit to initiate translation［J］. RNA, 2008, 14（1）：134-147.
［34］ ?oniewska-Lwowska A, Che?stowska S, Zagórski-Ostoja W, et al. Elements regulating Potato leafroll virus sgRNA1 translation are located within the coding sequences of the coat protein and read-through domain［J］. Acta Biochimica Polonica, 2009, 56（4）：619-625.
［35］Pogany J, Nagy PD. Authentic replication and recombination of Tomato bushy stunt virus RNA in a cell-free extract from yeast［J］. Journal of Virology, 2008, 82（12）：5967-5980.
［36］Pogany J, Stork J, Li ZH, et al. In vitro assembly of the Tomato bushy stunt virus replicase requires the host heat shock protein 70［J］. Proceedings of the National Academy of Sciences, 2008, 105（50）：19956-19961.
［37］Xu K. Huang TS, Nagy PD. Authentic in vitro replication of two tombusviruses in isolated mitochondrial and endoplasmic reticulum membranes［J］. Journal of Virology, 2012, 86（23）：12779-12794.
［38］Iki T, Yoshikawa M, Nishikiori M, et al. In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90［J］. Molecular Cell, 2010, 39（2）：282-291.
［39］Iwakawa HO, Tomari Y. Molecular insights into microRNA-mediated translational repression in plants［J］. Molecular Cell, 2013, 52（4）：591-601.
［40］Mercx S, Tollet J, Magy B, et al. Gene inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 suspension cells［J］. Frontiers in Plant Science, 2016, 7：40.

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[2]	韦雪芳;王冬梅;刘思;周鹏;. 信号肽及其在蛋白质表达中的应用[J]. , 2006, 0(06): 38-42.