| Description: | Our aim is to identify transcriptional changes occurs in Arabidopsis plants depleted of CK2 kinase activity. CK2 is a pleiotropic enzyme, essential for maintaining life, and with multiple isoforms. These facts have made important to use a dominant-negative mutant approach, by overexpressing a CK2 alfa kinase inactive subunit (CKA3mut), cloned downstream of the GVG inducible promoter (Moreno-Romero et al. 2008). This stably transformed CK2mut plants showed that longtime inductions of the transgene were lethal. However, short-time inductions were not lethal and revealed broad phenotypic changes in CK2 activity depleted plants, some of them related to defects in auxin transport (Moreno-Romero et al. 2008; Marques-Bueno et al. 2011). For the transcriptome analysis, the use of the inducible promoter made important to include in the experimental design control plants transformed with empty vector (only GVG receptor and with similar levels of GVG expression to CK2mut) for discarding side effects of the inducible system. METHOD: Arabidopsis seeds were surface sterilized, cold-treated at 4 C for 48 hours, and then sown on MS plates and grown for five days. Afterwards, plantlets were transferred to MS plates supplemented with 1uM Dex and grown for two days. Total RNA was isolated from frozen plantlets with Trizol (Invitrogen) and later puried using Qiagen RNA easy Plant Mini Kit columns (Qiagen, Hilden, Germany). RNA quality was checked using Experion (Bio-Rad laboratories). Three independent RNA preparations were made from CK2mut pooled samples and two from Empty vector samples (Moreno-Romero et al. 2011). Moreno-Romero et al. 2008. A role for protein kinase CK2 in plant development: evidence obtained using a dominant-negative mutant. The Plant Journal 55, 118-130 [2008].Moreno-Romero et al. 2011. About the role of CK2 in plant signal transduction. Molecular and Cellular Biochemistry 356(1-2):233-40.Marques-Bueno et al. 2011. a dominant negative mutant of protein kinase CK2 exhibits altered auxin responses in Arabidopsis. The Plant Journal 67, 169-180. |
NASCArrays Information at The BAR
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