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| Comparison of Effects of PolⅡ-derived Short RNA and RNA Silencing Suppressor on TMV-Based Expression Vector?System |
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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.
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Received: 26 May 2015
Published: 23 November 2015
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