| Description: | Maize knotted1 (kn1)and its homologs from different plant species are essential for meristem maintenance. In order to further understand their function, we are taking several approaches to identify target genes in Arabidopsis. We have made transgenic Arabidopsis plants that express a fused KN1 - glucocorticoid receptor (GR) protein. This fused protein is cytoplasmic in the absence of an inducer, but translocates to the nucleus upon induction with dexamethasone (Dex). Consequently, the plants show no phenotype without induction, but show the typical kn1 overexpression phenotype upon Dex induction. We propose to use the Arabidopsis EST microarray to compare 2 sets of RNAs from these plants: 1. mRNA of control, uninduced kn1-GR seedlings and Dex + cyclohexamide treated kn1-GR seedlings. We will induce kn1-GR seedlings with Dex but prevent protein translation by using cyclohexamide. The use of cyclohexamide prevents the expression of genes farther downstream of the kn1 signal, which is essential if kn1 induces the expression of other transcription factors. We will collect RNA from noninduced and induced seedlings 8 hours after induction. This time point was selected because immunolocalization experiments using a KN1 antibody showed clear nuclear localization of KN1 protein 8 hours after DEX induction. We predict that the mRNA population of induced seedlings will differ from that of non-induced control seedlings only in the expression of genes that are up- or down- regulated by kn1. 2. mRNA of control uninduced kn1-GR seedlings and uninduced kn1-GR leaves. Since the KN1-GR fusion product is expressed throughout the kn1-GR plants, we expect that some of the resulting genes, differentially expressed between non induced and Dex induced kn1-GR plants, will not be relevant to meristem function. In order to eliminate those before further characterization, we propose to use the microarray for this second set of mRNA. A comparison of the uninduced whole seedlings, control to leaves only of the same uninduced seedlings, will help us enrich for mRNAs relevant for meristem function. |
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