title: New generation of artificial MicroRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis.
abstract: Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are used for small RNA-based, specific gene silencing or knockdown in plants. Current methods to generate amiRNA or syn-tasiRNA constructs are not well adapted for cost-effective, large-scale production or for multiplexing to specifically suppress multiple targets. Here, we describe simple, fast, and cost-effective methods with high-throughput capability to generate amiRNA and multiplexed syn-tasiRNA constructs for efficient gene silencing in Arabidopsis (Arabidopsis thaliana) and other plant species. amiRNA or syn-tasiRNA inserts resulting from the annealing of two overlapping and partially complementary oligonucleotides are ligated directionally into a zero background BsaI/ccdB-based expression vector. BsaI/ccdB vectors for amiRNA or syn-tasiRNA cloning and expression contain a modified version of Arabidopsis MIR390a or TAS1c precursors, respectively, in which a fragment of the endogenous sequence was substituted by a ccdB cassette flanked by two BsaI sites. Several amiRNA and syn-tasiRNA sequences designed to target one or more endogenous genes were validated in transgenic plants that (1) exhibited the expected phenotypes predicted by loss of target gene function, (2) accumulated high levels of accurately processed amiRNAs or syn-tasiRNAs, and (3) had reduced levels of the corresponding target RNAs.
authors.affiliation: Donald Danforth Plant Science Center, St. Louis, Missouri 63132.
authors.forename: Alberto
authors.initials: A
authors.lastname: Carbonell
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takeda
authors.forename: Noah
authors.initials: N
authors.lastname: Fahlgren
authors.forename: Simon C
authors.initials: SC
authors.lastname: Johnson
authors.forename: Josh T
authors.initials: JT
authors.lastname: Cuperus
authors.forename: James C
authors.initials: JC
authors.lastname: Carrington
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D020868:Gene Silencing; D005822:Genetic Vectors; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D018749:RNA, Plant / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D018749:RNA, Plant; D034741:RNA, Small Interfering