A NEW BSMV-BASED VECTOR WITH MODIFIED ß MOLECULE ALLOWS SIMULTANEOUS AND STABLE SILENCING OF TWO GENES
Journal Title: Cellular and Molecular Biology Letters - Year 2012, Vol 17, Issue 1
Abstract
Virus-induced gene silencing is an important tool for functional gene analysis and the vector based on Barley stripe mosaic virus (BSMV) is widely used for the purpose in monocots. Of the tripartite BSMV genome, currently the BSMV:γMCS molecule is used to clone a fragment of a target gene. As an alternative, the BSMV:ß molecule was engineered with a unique BamHI site between the open reading frame of ßc (ORF ßc) and poly(A). The mixture of RNA particles α, ßBam HI and γ MCS was fully infectious. Barley phytoene desaturase and wheat phospholipase Dα fragments were cloned to ßBam HI and γ MCS. Delivery of the target gene fragment in γ MCS induced stronger silencing, while delivery in ßBam HI yielded more stable transcript reduction. A quantitative analysis (qRT-PCR) of the transcripts showed that the silencing induced with a fragment carried in both particles was stronger and more stable than that from a fragment placed in one particle. The modification of ß enables simultaneous silencing of two genes. Quantifying the ß and γ particles in virus-inoculated plants revealed a 2.5-fold higher level of γ than ß, while the stability of the insert was higher in ß compared with γ . The possible influence of the relative quantity of ß and γ particles in virus-inoculated plants on insert stability and gene silencing efficiency is discussed.
Authors and Affiliations
Adam Kawalek, Marta Dmochowska-Boguta, Anna Nadolska-Orczyk, Waclaw Orczyk
Keywords
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- EP ID EP86703
- DOI 10.2478/s11658-011-0041-9
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