title: Synthesis of an artificial Vitis vinifera miRNA 319e using overlapping long primers and its application for gene silencing.
abstract: The conserved mechanism of action of micro-RNAs (miRNAs) as regulators of gene expression has allowed the use of artificial miRNAs (amiRNAs) as a powerful tool for candidate gene evaluation in plants. Based on the use of a Vitis vinifera miRNA molecule (i.e., vvi-miR319e), the present work presents a new methodology for designing artificial miR319e precursors (pre-amiR319e). As a proof of concept, we silenced the green fluorescent protein (GFP) gene in transgenic Nicotiana benthamiana plants. This methodology includes a two-step PCR reaction in which overlapping long primers allow for the complete generation of pre-amiR319e-GFP molecules that are adequate for recombination into Gateway vectors with no further requirements. The seed region in amiRNA was directed against the 3'-end portion of the GFP gene. Three groups of transformed N. benthamiana plants were generated: GFP-, amiR319e-GFP-, and GFP plus miR319e-GFP-expressing vectors. A similar group of wild-type plants was included. Confocal microscopy evaluation of these groups revealed strong silencing of the GFP phenotype in the double GFP plus amiR319e-GFP group. The molecular characterization of silenced plants was achieved via modified 5'RACE of the GFP mRNA and revealed the occurrence of a partial, 3'-end GFP mRNA molecule that was generated in planta. In addition, large-scale small RNA sequencing confirmed the occurrence of the expected 21-nt miR319e-GFP species and other 22- and 24-nt species that exhibited sequence relationships with the expected amiRNA. These results highlight the possibility of using vvi-MIR319 as a template for the generation of single amiRNAs as a tool for gene silencing in plants.
authors.affiliation: Biotechnology Doctoral Program, Chemistry and Biological Sciences Faculty, Universidad de Santiago de Chile, Av. Libertador Bernardo O'Higgins 3363, Santiago, Chile.
authors.forename: Álvaro
authors.initials: Á
authors.lastname: Castro
authors.affiliation: Universidad Nacional Andrés Bello, Biological Sciences Faculty, Av. República 239, Santiago, Chile.
authors.forename: Daniela
authors.initials: D
authors.lastname: Quiroz
authors.affiliation: Biotechnology Laboratory, La Platina Research Station, Instituto de Investigaciones Agropecuarias, Santa Rosa 11610, La Pintana, Santiago, Chile.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Sánchez
authors.affiliation: Biotechnology Laboratory, La Platina Research Station, Instituto de Investigaciones Agropecuarias, Santa Rosa 11610, La Pintana, Santiago, Chile.
authors.forename: María de Los Ángeles
authors.initials: Mde L
authors.lastname: Miccono
authors.affiliation: Biotechnology Laboratory, La Platina Research Station, Instituto de Investigaciones Agropecuarias, Santa Rosa 11610, La Pintana, Santiago, Chile.
authors.forename: Carlos
authors.initials: C
authors.lastname: Aguirre
authors.affiliation: Chemistry and Biological Sciences Faculty, Universidad de Santiago de Chile, Av. Libertador Bernardo O'Higgins 3363, Santiago, Chile.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Ramírez
authors.affiliation: Biotechnology Laboratory, La Platina Research Station, Instituto de Investigaciones Agropecuarias, Santa Rosa 11610, La Pintana, Santiago, Chile.
authors.forename: Christian
authors.initials: C
authors.lastname: Montes
authors.affiliation: Biotechnology Laboratory, La Platina Research Station, Instituto de Investigaciones Agropecuarias, Santa Rosa 11610, La Pintana, Santiago, Chile. Electronic address: hprieto@inia.cl.
authors.forename: Humberto
authors.initials: H
authors.lastname: Prieto
mesh_terms: D017931:DNA Primers / Q000235:genetics*; D020868:Gene Silencing; D035683:MicroRNAs / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics* / Q000378:metabolism; D027843:Vitis / Q000235:genetics*
chemical_list: D017931:DNA Primers; D035683:MicroRNAs; D018749:RNA, Plant
keywords: Artificial microRNA; Gene silencing; Small RNA sequencing; Vitis vinifera; amiRNA synthesis