title: Vectors and methods for hairpin RNA and artificial microRNA-mediated gene silencing in plants.
abstract: In plant cells, DICER-LIKE4 processes perfectly double-stranded RNA (dsRNA) into short interfering (si) RNAs, and DICER-LIKE1 generates micro (mi) RNAs from primary miRNA transcripts (pri-miRNA) that form fold-back structures of imperfectly dsRNA. Both si and miRNAs direct the endogenous endonuclease, ARGONAUTE1 to cleave complementary target single-stranded RNAs and either small RNA (sRNA)-directed pathway can be harnessed to silence genes in plants. A routine way of inducing and directing RNA silencing by siRNAs is to express self-complementary single-stranded hairpin RNA (hpRNA), in which the duplexed region has the same sequence as part of the target gene's mRNA. Artificial miRNA (amiRNA)-mediated silencing uses an endogenous pri-miRNA, in which the original miRNA/miRNA* sequence has been replaced with a sequence complementary to the new target gene. In this chapter, we describe the plasmid vector systems routinely used by our research group for the generation of either hpRNA-derived siRNAs or amiRNAs.
authors.affiliation: School of Microbial and Molecular Sciences, University of Sydney, Sydney, Australia. a.eamens@usyd.edu.au
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Eamens
authors.forename: Peter M
authors.initials: PM
authors.lastname: Waterhouse
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D005822:Genetic Vectors; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D010944:Plants / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D035683:MicroRNAs; D012330:RNA, Double-Stranded; D018749:RNA, Plant; D034741:RNA, Small Interfering