title: A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants.
abstract: The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids) and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana. We assembled an RNA interference (RNAi) module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP) and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct.
authors.affiliation: Faculty of Biology, Genetics, University of Munich (LMU), Martinsried, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Binder
authors.affiliation: Faculty of Biology, Genetics, University of Munich (LMU), Martinsried, Germany.
authors.forename: Jayne
authors.initials: J
authors.lastname: Lambert
authors.affiliation: Faculty of Biology, Genetics, University of Munich (LMU), Martinsried, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Morbitzer
authors.affiliation: Faculty of Biology, Genetics, University of Munich (LMU), Martinsried, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Popp
authors.affiliation: Faculty of Biology, Genetics, University of Munich (LMU), Martinsried, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ott
authors.affiliation: Faculty of Biology, Genetics, University of Munich (LMU), Martinsried, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lahaye
authors.affiliation: Faculty of Biology, Genetics, University of Munich (LMU), Martinsried, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Parniske
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D005816:Genetic Complementation Test; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins; D007438:Introns; D000070116:Lotus / Q000235:genetics; D010641:Phenotype; D030821:Plants, Genetically Modified / Q000235:genetics; D010957:Plasmids; D014026:Nicotiana / Q000235:genetics; D019076:Transgenes
chemical_list: D004269:DNA, Bacterial; C036483:T-DNA; D049452:Green Fluorescent Proteins