7种RNA沉默抑制子对植物病毒载体表达系统表达水平的影响

摘要/Abstract

摘要： 目的探索不同植物病毒RNA沉默抑制子对植物病毒载体表达系统重组蛋白表达水平的作用，为合理高效地利用这一新
型的外源基因表达平台奠定基础。方法构建了7种不同的RNA沉默抑制子瞬时表达载体，以农杆菌渗滤法，与马铃薯X病毒
表达载体PVXdt-GFP共侵染寄主植物本明烟，通过对报告基因绿色荧光蛋白（GFP）的荧光观察，并以Western blotting、ELISA
和RT-qPCR等测定GFP在烟草中的表达情况，分析不同RNA沉默抑制子对植物中外源基因表达水平的作用和特点。结果7
种病毒RNA沉默抑制子对外源基因GFP在烟草中表达水平的作用效果和持续时间存在差异，其中，番茄丛矮病毒的P19蛋白
的增效作用最好，作用时间也最长；非洲木薯花叶病毒的AC2蛋白和水稻黄斑驳病毒的P1蛋白无明显的增效作用。结论
RNA沉默抑制子可以通过抑制病毒诱导的RNA沉默来提高外源基因在植物中的表达水平和表达持续时间，但是不同的植物病
毒载体表达系统需要通过筛选获得其最佳的共表达沉默抑制子“伴侣”。

Abstract: ObjectiveTo test the effects of7virus-encoded RNA silencing suppressors (RSSs) for enhancement of a plant
virus-based vector system-mediated heterologous expression of green fluorescence protein (GFP) inNicotiana benthamiana.
MethodsSeven transient expression vectors for the7RSSs were constructed and co-inoculated on the leaves ofNicotiana
benthamiana with PVXdt-GFP vector, a novelPotato virusX-based plant expression vector, through agroinfiltration. The
protein and mRNA expression levels of the reporter gene GFP in the co-inoculatedNicotianaleaves were examined by Western
blotting, ELISA and RT-qPCR to assess the effect of the RSSs for GFP expression enhancement.ResultsThe7RSSs differed in
the degree and duration of enhancement of heterologous GFP expression, and the p19protein ofTomato bushy stunt virus
(TBSV) induced the highest expression of GFP. African cassava mosaic virusAC2protein andRice yellow mettle virusP1
protein produced no obvious enhancement GFP expression.ConclusionTransient co-expression of RSSs suppresses host
silencing response to allow high-level and long-term expression of heterologous genes in plant, but the optimal RSS has to be
identified for each plant virus-based expression vector system.

王盛，董洁，曹慜，穆红珍，丁国平，张虹. 7种RNA沉默抑制子对植物病毒载体表达系统表达水平的影响[J]. 南方医科大学学报, 2012, 32(11): 1536-.

参考文献

［1］Boehm R. Bioproduction of therapeutic proteins in the21st centuryand the role of plants and plant cells as production platforms［J］.
Ann N Y Acad Sci,2007,1102:121-34.
［2］Lico C, Chen Q, Santi L. Viral vectors for production of recom-binant proteins in plants［J］. J Cell Physiol, 2008,216:366-77.
［3］Gleba Y, Klimyuk V, Marillonnet S. Viral vectors for the expression of proteins in plants［J］. Curr Opin Biotechnol,2007,18(2):134-41.
［4］Scholthof HB. Heterologous expression of viral RNA interference suppressors: RISC management［J］. Plant Physiol, 2007, 145(4):1110-7.
［5］Voinnet O. Induction and suppression of RNA silencing: insights from viral infections ［J］. Nat Rev Genet,2005,6(3):206-20.
［6］Johansen LK, Carrington JC. Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system ［J］. Plant Physiol,2001,126(3):930-8.
［7］Voinnet O, Rivas S, Mestre P, et al. An enhanced transient expression system in plants based on suppression of gene silencing by the p19protein of tomato bushy stunt virus［J］. Plant J, 2003, 33 (5):949-56.
［8］Sainsbury F, Lomonossoff GP. Extremely high-level and rapidtransient protein production in plants without the use of viral replication ［J］. Plant Physiol,2008,148(3):1212-8.
［9］Burgyán J, Havelda Z. Viral suppressors of RNA silencing［J］.Trends Plant Sci,2011,16(5):265-72.
［10］Komarova TV, Skulachev MV, Zvereva AS, et al. New viral vector for efficient production of target proteins in plants ［J］. Biochemistry (Mosc),2006,71(8):846-50.
［11］Li H, Li WX, Ding SW. Induction and suppression of RNA silencing by an animal virus ［J］. Science,2002,296:1319-21.
［12］Dong X, van Wezel R, Stanley J, et al. Functional characterization of the nuclear localization signal for a suppressor of posttranscriptional gene silencing［J］. J Virol,2003,77(12):7026-33.
［13］Voinnet O, Pinto YM, Baulcombe DC. Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants［J］. Proc Natl Acad Sci U S A, 1999,96(24):14147-52.
［14］Voinnet O, Lederer C, Baulcombe DC. A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana［J］. Cell,2000,103(1):157-67.
［15］Reed JC, Kasschau KD, Prokhnevsky AI, et al. Suppressor of RNA silencing encoded by Beet yellows virus［J］. Virology, 2003, 306(2):203-9.
［16］Ye K, Patel DJ. RNA silencing suppressor p21of Beet yellows virus forms an RNA binding octameric ring structure［J］. Structure, 2005,13(9):1375-84.
［17］Qu F, Morris TJ. Efficient infection of Nicotiana benthamiana by Tomato bushy stunt virus is facilitated by the coat protein and maintained by p19through suppression of gene silencing［J］. Mol Plant Microbe Interact,2002,15(3):193-202.
［18］Lacombe S, Bangratz M, Vignols F, et al. The rice yellow mottle virus P1protein exhibits dual functions to suppress and activate gene silencing［J］. Plant J,2010,61(3):371-82.
［19］Anandalakshmi R, Gj P, Ge X, et al. A viral suppressor of gene silencing in plants ［J］. Proc Natl Acad Sci USA, 1998,95:13079-84.
［20］Grimsley N, Hohn B, Hohn T, et al. "Agroinfection," an alternative route for viral infection of plants by using the Ti plasmid［J］. Proc Natl Acad Sci USA,1986,83(10):3282-6.
［21］Velásquez AC, Chakravarthy S, Martin GB. Virus-induced gene silencing (VIGS) in Nicotiana benthamiana and tomato［J］. J Vis Exp,2009(28):1-4.
［22］Ma P, Liu J, He H, et al. A viral suppressor P1/HC-pro increases the GFP gene expression in agrobacterium-mediated transient assay［J］. Appl Biochem Biotechnol,2009,158(2):243-52.
［23］Streatfield SJ. Approaches to achieve high-level heterologous pro-tein production in plants ［J］. Plant Biotechnol J,2007,5(1):2-15.
［24］Saxena P, Hsieh YC, Alvarado VY, et al. Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless［J］. Plant Biotechnol J,2011,9(6):703-12.
［25］Chiba M, Reed JC, Prokhnevsky AI, et al. Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon［J］. Virology,2006,346(1):7-14.
［26］Lindbo JA. TRBO: a high-efficiency tobacco Mosaic virus RNA-based overexpression vector［J］. Plant Physiol, 2007, 145(4):1232-40.