IBONE   05434
Unidad Ejecutora - UE
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Visit to EMBRAPA CENARGEN LABORATORY, Brasilia, Brazil, under an international collaboration with Dr. Vera Tavares de Campos Carneiro supported by PROSUL. Project: ANÁLISE COMPARATIVA DO PERFIL DE EXPRESSÃO GÊNICA DA REPRODUÇÃO POR APOMIXIA NAS FORRAGEIRAS PASPALUM E BRACHIARIA. OBJECTIVES: 1) Compare the pattern of spatial and temporal expression of two genes (B22/B137/N46 and B61/N108) previously associated with the development of apomixis in both Brachiaria brizantha and Paspalum notatum. 2) Produce a set of Arabidopsis thaliana plants with altered expression for selected candidates. 3) Analyze Arabidopsis thaliana loss of function mutant plants involving selected candidates. EXPERIMENTAL ACTIVITIES: 1) Analysis of gene expression by RT‐qPCR and in situ hybridization of Brachiaria reproductive tissue. 2) Transformation of Arabidopsis thaliana plants with constructs including the genes of interest. 3) Morphological characterization of mutants of A. SALK_044117C and SALK_203147C thaliana. METHODOLOGY: 1) RNA isolation and cDNA synthesis: RNA was extracted from ovaries and flowers separately. RNA was isolated from about 100 mg of fresh tissue using Qiagen RNeasy Plant Mini Kit. cDNA samples were prepared from 0,5 μg of total RNA and Oligo-dT primer using the SuperScript III Reverse Transcriptase enzyme. 2) Real time PCR: PCR reactions were performed in Mastercyclereprealplex (EppendorfTM). Three biological replicates were performed for each sample. Relative expression of genes at different reproductive developmental stages was estimated by the 2‐ddCt method. 3) In situ hybridization: In situ hybridization was performed in sections of 3.5 μm of ovaries. Tissue preparation, hybridization, and posthybridization reactions were performed according to Dusi (2001). The RNA probe was synthesized using the Digoxigenin RNA labeling kit (Roche) . 4) Gateway cloning: Recombination reactions were performed for obtaining plant expression plasmids including the genes of interest. 5) Transformation of bacterial strains: All ligation products including the Gateway cassettes were used for transforming E. coli DB301 cells (Invitrogen), which are resistant to the ccdB gene. A. tumefaciens strain GV3101 was then transformed with recombinant plasmids for subsequent plant transformation. 6) Arabidopsis transformation: A. tumefaciens mediated transformation of A. thaliana ecotype Columbia was accomplished by using the floral dip technique. 7) Morphological analysis of mutant plants: Mutant plants were photographed and flowers from wild type and mutant plants were clarified by two different methods.