title: A simplified method for constructing artificial microRNAs based on the osa-MIR528 precursor.
abstract: Artificial miRNAs (amiRNAs) have been used successfully in various plants to silence endogenous genes or viral RNAs. The method of Schwab et al., widely used to construct amiRNAs, requires four PCRs. We describe a simplified method of constructing amiRNA based on the osa-MIR528 backbone using one PCR step by addition of prolonging sequence to the primers. The length of prolonging sequence needed in the osa-MIR528 precursor was determined. Using this method, we constructed amiRNA targeting the Nicotiana benthamiana UPF1 gene and showed that it functioned in silencing UPF1 expression in leaves when expressed transiently.
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Yan
authors.forename: Yuwen
authors.initials: Y
authors.lastname: Lu
authors.forename: Gentu
authors.initials: G
authors.lastname: Wu
authors.forename: Jiejun
authors.initials: J
authors.lastname: Peng
authors.forename: Hongying
authors.initials: H
authors.lastname: Zheng
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.forename: Jianping
authors.initials: J
authors.lastname: Chen
mesh_terms: D004268:DNA-Binding Proteins / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics*; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D018749:RNA, Plant / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D035683:MicroRNAs; D018749:RNA, Plant; C117995:osa protein, Drosophila