microRNA Plasmids
Donated by
- Javier Paz-Ares Departamento de Genetica Molecular de Plantas, Centro Nacional de Biotecnologia (CNB) - CSIC
- Detlef Weigel Department of Molecular Biology, Max-Planck-Institute for Developmental Biology
- Ignacio Rubio Somoza Department of Molecular Biology, Max-Planck-Institute for Developmental Biology
- Marco Todesco Department of Molecular Biology, Max-Planck-Institute for Developmental Biology
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Description
microRNA Plasmids
Knockdowns achieved by using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation.
Abstract
Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for ~20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA–resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.
microRNA clones held by NASC
As these microRNA plasmids are based on pGREEN (www.pgreen.ac.uk) they will only replicate in Agrobacterium containing the pSOUP plasmid. An aliquot of pSOUP will be included with all clone orders. pSOUP confers tetracycline (10mg/L) resistance in bacteria.
pSOUP/pGreen vectors can be transformed into Escherichia coli strain DH5a and also the Agrobacterium strains LBA4404, GV2260, GV3600, EHA105, A GL-1, LBA9402.
Please refer EMBL/Genbank record AJ007829 for pGREEN sequence information. For MIM construct primers used please see Table S1 in Todesco et al., 2010.
Selection
The donors of the microRNA plasmids state that they used a modified version of the selection marker in which the kanamycin-resistance gene has been replaced with spectinomycin. Use spectinomycin (50 mg/L working concentration) for selection in bacteria, BASTA for selection in plants.
Related links
References
- Hellens, R.P. et al. 2000. pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Molecular Biology 42(6): 819-32. PMID. 10890530
- Todesco, M. et al. 2010. A Collection of Target Mimics for Comprehensive Analysis of MicroRNA Function in Arabidopsis thaliana. PLoS Genetics 6(7): e1001031. PMC. 2908682