Rapid induction of transcriptional and post-transcriptional gene silencing using a novel Cucumber mosaic virus vector

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We developed a novel RNA virus vector based on the Cucumber mosaic virus (CMV), which is able to efficiently induce gene silencing in plants. We manipulated the RNA 2 of the CMV Y strain, whose genome consists of tripartite components, and introduced restriction sites for cloning a foreign sequence into the vector. To evaluate the vector (designated CMV2-A1) in terms of the ability to induce gene silencing, we cloned portions of the green fluorescent protein (GFP) cDNA or Cauliflower mosaic virus (CaMV) 35S promoter sequences into the vector and inoculated the infectious transcripts into Nicotiana benthamiana plants that express the GFP gene under the control of the CaMV 35S promoter. In both cases, a loss of GFP fluorescence accompanying a reduction in the level of GFP mRNA was induced. The short interfering RNAs (siRNAs) harboring the sequences inserted in the CMV2-A1 vector were detected in the silenced plants. When plants were infected with the virus containing the CaMV 35S promoter sequence, the CaMV 35S promoter sequence in the genomic DNA was heavily methylated. A reduction in the mRNA level of the GFP gene and loss of GFP fluorescence were induced as early as 6 and 12 days post-inoculation, respectively, earlier than the 20 days previously achieved with a Potato virus X vector. These results suggest that the CMV2-A1 vector is suitable for the rapid induction of both transcriptional and post-transcriptional gene silencing.

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