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Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 10 No. 2, Issue of January 15, 2007
© 2007 by Pontificia Universidad Católica de Valparaíso -- Chile Received August 23, 2006 / Accepted November 9, 2006
DOI: 10.2225/vol10-issue2-fulltext-11
RESEARCH ARTICLE

A practical approach to the understanding and teaching of RNA silencing in plants

Ariel A. Bazzini
Instituto Nacional de Tecnología Agropecuaria
Instituto de Biotecnología
Las Cabañas y Los Reseros
B1712WAA Castelar, Argentina
Tel: 5411 4621 1447. Ext 146
Fax: 5411 4621 0199
E-mail: abazzini@cnia.inta.gov.ar 

Vanesa C. Mongelli
Instituto Nacional de Tecnología Agropecuaria
Instituto de Biotecnología
Las Cabañas y Los Reseros
B1712WAA Castelar, Argentina
Tel: 5411 4621 1447. Ext 146
Fax: 5411 4621 0199
E-mail: vmongeli@cnia.inta.gov.ar

H. Esteban Hopp
Unidad Integrada de Investigación y Docencia
Centro Nacional de Investigaciones Agropecuarias
Facultad de Ciencias Exactas y Naturales
Universidad de Buenos Aires
Las Cabañas y Los Reseros,
B1712WAA Castelar, Argentina
Tel: 5411 4621 1447. Ext 146
Fax: 5411 4621 0199
E-mail: ehopp@cnia.inta.gov.ar

Mariana del Vas
Instituto de Tecnología Agropecuaria
Instituto de Biotecnología
Las Cabañas y Los Reseros,
B1712WAA Castelar, Argentina
Tel: 5411 4621 1447. Ext 146
Fax: 5411 4621 0199
E-mail: mdelvas@cnia.inta.gov.ar

Sebastián Asurmendi*
Instituto de Tecnología Agropecuaria
Instituto de Biotecnología
Las Cabañas y Los Reseros
B1712WAA Castelar, Argentina
Tel: 5411 4621 1447. Ext 146
Fax: 5411 4621 0199
E-mail: sasurmendi@cnia.inta.gov.ar

*Corresponding author

Financial support: This research was partially supported by grants PICT 11196, PID 267 and PAV 137 from FONCyT-ANPCyT and PIP 5788 from CONICET and other funding from INTA, Argentina.

Keywords: gene silencing, GFP, HC-Pro, PDS, protocols, PTGS, teaching, transgenic, transient expression, TRV, VIGS.

Abbreviations:

dsRNA: double stranded RNA
gfp: green fluorescent protein
PDS: phytoene desaturase
PTGS: Post-transcriptional gene silencing
RNAi: RNA interference
siRNA: short interfering RNAs
TRV: Tobacco rattle virus

Abstract   Full Text

Gene silencing, also called RNA interference (RNAi) is a specific mechanism of RNA degradation involved in gene regulation, development and defense in eukaryotic organisms. It became an important subject in the teaching programs of molecular biology, genetics and biotechnology courses in the last years. The aim of this work is to provide simple and inexpensive assays to understand and teach gene silencing using plants as model systems. The use of transient and permanent transgenic plants for expressing reporter genes, like those derived from jellyfish green fluorescent protein (gfp) encoding gene, provides a nice, colorful and conclusive image of gene silencing. Three experimental approaches to evidence RNA silencing are depicted. In the first approach gene silencing is demonstrated after transient expression of reporter genes in non-transgenic plants. In the second, silencing is triggered against a reporter gene stably integrated into a transgenic plant. The third approach involves the triggering of RNA silencing against endogenous genes using viral vectors. In addition we illustrate systemic gene silencing showing how the silencing signal is spread over a plant and finally it is also demonstrated the suppression of gene silencing. The first group of experiments is recommended to be tough on undergraduate courses, the following two sections are recommended for graduate courses. Hopefully, it will help students to understand this important phenomenon and to unravel the importance of gene silencing as a key gene regulation mechanism and as a molecular and biotechnological tool.

Supported by UNESCO / MIRCEN network