聚合酶Ⅱ转录的sRNA与RNA沉默抑制子对TMV病毒载体表达系统作用的比较

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摘要聚合酶Ⅱ介导合成的外源短链RNA(short RNA，sRNA)可以竞争性地抑制内源mRNA的核质转运，推测共表达sRNA可以间接地提高病毒载体表达系统的表达效率。为了验证这一假说和评价其应用潜能，本研究采用基因体外合成技术和亚克隆技术，分别构建了聚合酶Ⅱ转录的拟南芥U6-1 RNA和黄瓜花叶病毒(Cucumber mosaic virus, CMV)编码的RNA沉默抑制子(RNA silencing suppressors, RSSs)2b的植物表达载体。以农杆菌渗滤技术，与烟草花叶病毒(Tobacco mosaic virus, TMV)表达载体共接种寄主植物本氏烟(Nicotiana benthamiana)，通过对报告基因绿色荧光蛋白(green fluorescence protein, GFP)的荧光观察，并以Western blot和酶联免疫吸附测定法(enzyme-linked?immunosorbnent?assay, ELISA)测定GFP在烟草中的表达情况，分析sRNA对植物病毒载体表达系统的作用和可能的作用机制。同时通过与RSSs比较，评价sRNA在植物生物反应器中的应用潜能。实验结果表明，聚合酶Ⅱ转录的sRNA能有效地提高TMV病毒载体介导的外源基因在植物中的表达(GFP表达量比对照组高约2.5倍)，而且能够显著地降低TMV诱导的病程相关蛋白2(Pathogenesis-related protein 2, PR2)在细胞内的积累水平(PR2表达量比对照组下降约8倍)。推测Ⅱ型启动子转录的sRNA通过竞争性地抑制宿主抵御病毒入侵相关蛋白合成的mRNA核质运转促进病毒RNA介导的外源基因在植物中的表达。此外，Ⅱ型启动子转录的sRNA的作用效果与CMV病毒编码的RNA沉默抑制子2b相当。研究结果为提高病毒载体介导的外源基因在植物中的表达提供了一条可供借鉴的思路。

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	马婷
	张西倩
	丁向真
	李志英
	王盛

关键词 ： RNA聚合酶Ⅱ, 烟草花叶病毒(TMV), 表达载体, U6核内小RNA, RNA转运

Abstract：PolⅡ promoter-derived short RNAs can interfere with cellular mRNA for nuclear export and may create conditions for enhancing heterologous gene expression mediated by plant virus RNA-based vectors in plant. To test this hypothesis, we synthesized Arabidopsis thaliana U6-1 RNA gene and the Cucumber mosaic virus (CMV)-encoded silencing suppressor protein 2b gene in vitro and constructed PolⅡpromoter-directed U6 RNA and CMV-2b plant expression vector respectively. The vectors were co-inoculated on Nicotiana benthamiana plants with Tobacco mosaic virus (TMV)-based plant expression vector through agroinfiltration. The expression levels of green fluorescence protein (GFP) in inoculated Nicotiana leaves were then examined by Western blotting and ELISA respectively. All data were collected for the assessment of the effect of PolⅡ promoter-synthesized sRNA on plant virus RNA-based expression vector system. The results showed that expression of GFP mediated by TMV-based expression vector was significantly enhanced in the presence of PolⅡ promoter-derived short RNAs (approximately 2.5-fold higher than the?control levels). And the accumulation of pathogenesis-related protein 2 (PR2) induced by TMV-based vector was dramatically decreased in the presence of PolⅡ promoter-derived short RNAs (approximately eight times?lower than?the?control levels). We speculated that the competition of nuclear export resulted in decreased protective antiviral mRNAs export into the cytoplasm and so far enhanced GFP gene expression in the infiltrated plant tissues. Furthermore, co-expression with PolⅡ promoter-derived sRNA also significantly increased the expression of GFP to levels similar to those induced by CMV-2b suppressor. The results provides an alternative to improve recombinant protein expression in plants from agroinfection-compatible plant virus expression vectors.

Key words： RNA Polymerase Ⅱ Tobacco mosaic virus (TMV) Expression Vectors U6 snRNA Nuclear Export

收稿日期: 2015-05-26 出版日期: 2015-11-23

基金资助:利用TMV 在烟草中高效表达rPA（Reteplase）

通讯作者: 王盛 E-mail: wang_s@nxu.edu.cn

引用本文:

马婷张西倩丁向真李志英王盛. 聚合酶Ⅱ转录的sRNA与RNA沉默抑制子对TMV病毒载体表达系统作用的比较 [J]. , 2016, 24(1): 1-9.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I1/1

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