A new BSMV-based vector with modified beta molecule allows simultaneous and stable silencing of two genes

作者：Kawalek Adam; Dmochowska Boguta Marta; Nadolska Orczyk Anna; Orczyk Waclaw^*

来源：Cellular and Molecular Biology Letters, 2012, 17(1): 107-123.

DOI：10.2478/s11658-011-0041-9

• BSMV
• Cereals
• Functional analysis
• Phospholipase D
• Phytoene desaturase
• Silencing
• Wheat
• Vector stability
• VIGS

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:gamma MCS molecule is used to clone a fragment of a target gene. As an alternative, the BSMV:beta molecule was engineered with a unique BamHI site between the open reading frame of beta c (ORF beta c) and poly(A). The mixture of RNA particles alpha, beta BamHI and gamma MCS was fully infectious. Barley phytoene desaturase and wheat phospholipase D alpha fragments were cloned to beta BamHI and gamma MCS. Delivery of the target gene fragment in gamma MCS induced stronger silencing, while delivery in beta BamHI 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 beta enables simultaneous silencing of two genes. Quantifying the beta and gamma particles in virus-inoculated plants revealed a 2.5-fold higher level of gamma than beta, while the stability of the insert was higher in beta compared with gamma. The possible influence of the relative quantity of beta and gamma particles in virus-inoculated plants on insert stability and gene silencing efficiency is discussed.

• 出版日期2012-3