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Title:   Author  item
                                            Verdonk, Julianitem
                                            Sullivan, Michael
  Submitted to: Botany
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 10/29/2012
                                    Publication  Date: 1/1/2013
                                    Citation: Verdonk, J.C., Sullivan, M.L. 2013. Artificial micro RNA (amiRNA) induced gene silencing in alfalfa (Medicago sativa). Botany. 91:117-122.Interpretive Summary: Gene silencing is an effective tool to study the function of genes and the proteins they encode. Approaches like transcriptional profiling or proteomics to investigate plant biology often lead to the identification of novel genes or proteins with a hypothetical or unknown function. Even for genes or proteins with a ‘known’ function, it can be hard to predict what their exact role is in living plants. Recent advances in the model species Arabidopsis and rice have led to the development of a gene-silencing approach utilizing artificial micro-RNA (amiRNA). This approach can provide advantages over previously used methods, such as increased specificity (that is, silencing only the gene of interest and not similar gene family members). In addition, it requires simpler preparation of the DNA construct to carry out the silencing experiment. Although amiRNA silencing has been shown to work for the model species Arabidopsis and rice, there are no reports of its use in alfalfa (Medicago sativa).
   
Here we show that the amiRNA does work in alfalfa. This was demonstrated by silencing the expression of a beta-glucuronidase (GUS) gene that had previously been transformed into the alfalfa test plants. Expression of this transgene can easily be monitored by tissue staining and activity assays. Our findings show that amiRNA is an effective approach to gene silencing in alfalfa using already available web-based construct design tools developed for Arabidopsis. The ability to use this approach will provide researchers additional flexibility to study the functions of specific genes in alfalfa and most likely other closely related legume species. Consequently, the work reported here will be of interest to scientists using molecular biology approaches to study various aspects of plant biology and physiology in these species.
                                Technical Abstract: Gene silencing is a powerful technique that allows the study of the function of specific genes by selectively reducing their transcription. Several different approaches can be used; however, they all have in common the artificial generation of single-stranded small RNAs that are utilized by the endogenous gene silencing machinery of the organism. Artificial micro RNAs (amiRNA) can be used to specifically target genes for silencing because only a short sequence of 21 nucleotides of the gene of interest is used. Gene silencing via amiRNA has been developed for Arabidopsis thaliana and rice using endogenous miRNA precursors. It has been shown to also work effectively in other dicot species using the arabidopsis miRNA precursor. Here, we demonstrated that the arabidopsis miR319 precursor can be used to silence genes in the important forage crop species alfalfa (Medicago sativa L.) by silencing the expression of a transgenic beta-glucuronidase (GUSPlus) target gene.


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