Synthesis of an artificial Vitis vinifera miRNA 319e using overlapping long primers and its application for gene silencing

• Published in: Journal of biotechnology 2016-09, Vol.233, p.200-210
• Main Authors: Castro, Álvaro, Quiroz, Daniela, Sánchez, Evelyn, Miccono, María de los Ángeles, Aguirre, Carlos, Ramírez, Alejandra, Montes, Christian, Prieto, Humberto
• Format: Article
• Language: English
• Subjects: amiRNA synthesis; Artificial microRNA; DNA Primers - genetics; Gene expression; Gene sequencing; Gene Silencing; Genes; Mathematical analysis; Methodology; MicroRNAs - genetics; Nicotiana benthamiana; Plants (organisms); Ribonucleic acids; RNA, Plant - genetics; RNA, Plant - metabolism; Small RNA sequencing; Vectors (mathematics); Vitis - genetics; Vitis vinifera
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2016.06.028

•A two-step PCR for synthesize an artificial Vitis vinifera microRNA is described.•After synthesis the generated pre-microRNA contains adequate recombination signals.•Confocal analyses showed strong silencing of the GFP in stable transgenic plants.•Small RNA sequencing evidenced the occurrence of expected species.•Results support the use of using vvi-MIR319 as a tool for gene silencing in plants. 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.