Identificação e caracterização de promotores de genes de café (coffea arabica)
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Data
2009-12-15
Autores
Cavallari, Carla Fernanda Barsalobres [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A caracterização de promotores ubíquos, tecido-específicos ou induzidos sob determinada condição é importante para a produção e liberação comercial de plantas geneticamente modificadas. Este trabalho apresenta a identificação e caracterização de promotores de genes de café (Coffea arabica), uma cultura de grande importância sócio-econômica. Foram selecionados 41 genes candidatos com padrão de expressão constitutivo, fruto-específico ou relacionados com o mecanismo de defesa, através de dados da literatura e da análise in silico em bancos de ESTs disponíveis. A expressão constitutiva ou específica foi confirmada através de PCR quantitativa para somente 10 dos genes analizados. Os experimentos foram realizados em 5 diferentes tecidos/órgãos (raiz, caule, folha, flor e fruto) ou em plantas tratadas com o fungo biotrófico Hemileia vastatrix. Regiões promotoras de 4 genes foram isoladas através de genome walking: CaGAPDH (glyceraldehyde-3-phosphate dehydrogenase como candidato constitutivo), CaAN2 (anthocyanin 2 específico de fruto), CaPR1b e CaNPR1 (basic form of Pathogenesis-Related protein type 1 e Nonexpressor of PR genes, correspondentes a genes induzidos mediante a presença de agentes patogênicos). As regiões cis-reguladoras foram caracterizadas in silico e a funcionalidade dos promotores foi avaliada in planta através da expressão do gene repórter uidA em experimentos de transformação transiente em plantas de tabaco ou tomate. Além do interesse fundamental para a compreensão dos mecanismos de regulação gênica em eucariotos, os resultados desta pesquisa são importantes para o desenvolvimento de plantas transgênicas, apresentando também potencial em programas de melhoramento genético do cafeeiro. Palavras-chaves: Coffea arabica, expressão gênica, promotores, genes ubíquos, frutos, resistência de plantas
The choice of promoter, to confer constitutive, spatial and/or temporal transgene expression, is important in plant biotechnology applications. This study presents the identification and characterization of different gene promoters in coffee (Coffea arabica), a species of major socioeconomic characteristics. Forty one constitutive, fruit-specific or pathogen defense-related genes were identified following literature data or in silico analyses in available EST databases. The expression levels of these genes were verified by quantitative PCR and only 10 genes presented a constitutive or specific expression condition. The expression levels assays were carried out in 5 coffee organs/tissues (root, stem, leaves, flowers and fruits) or in coffee plants challenged with Hemileia vastatrix. The promoter region of 4 genes were isolated and characterized in silico: CaGAPDH (glyceraldehyde- 3-phosphate dehydrogenase as inner control), CaAN2 (anthocyanin 2 fruitspecific), CaPR1b and CaNPR1 (basic form of Pathogenesis-Related protein type 1 and Nonexpressor of PR genes, both pathogen-inducible). The functional analyses of the DNA sequence upstream of these genes were assessed with regard to the uidA reporter gene, via Agrobacterium-mediated transient expression assay in tobacco or tomato plants. The characterization of promoters is not only an approach towards coffee breeding programs, but also provides fundamental data for understanding the mechanisms regulating gene expression in perennial plants
The choice of promoter, to confer constitutive, spatial and/or temporal transgene expression, is important in plant biotechnology applications. This study presents the identification and characterization of different gene promoters in coffee (Coffea arabica), a species of major socioeconomic characteristics. Forty one constitutive, fruit-specific or pathogen defense-related genes were identified following literature data or in silico analyses in available EST databases. The expression levels of these genes were verified by quantitative PCR and only 10 genes presented a constitutive or specific expression condition. The expression levels assays were carried out in 5 coffee organs/tissues (root, stem, leaves, flowers and fruits) or in coffee plants challenged with Hemileia vastatrix. The promoter region of 4 genes were isolated and characterized in silico: CaGAPDH (glyceraldehyde- 3-phosphate dehydrogenase as inner control), CaAN2 (anthocyanin 2 fruitspecific), CaPR1b and CaNPR1 (basic form of Pathogenesis-Related protein type 1 and Nonexpressor of PR genes, both pathogen-inducible). The functional analyses of the DNA sequence upstream of these genes were assessed with regard to the uidA reporter gene, via Agrobacterium-mediated transient expression assay in tobacco or tomato plants. The characterization of promoters is not only an approach towards coffee breeding programs, but also provides fundamental data for understanding the mechanisms regulating gene expression in perennial plants
Descrição
Palavras-chave
Café - melhoramento genético, Café, Housekeeping genes
Como citar
CAVALLARI, Carla Fernanda Barsalobres. Identificação e caracterização de promotores de genes de café (coffea arabica). 2009. 331 f. Dissertação (mestrado) - Universidade Estadual Paulista, Instituto de Biociências de Botucatu, 2009.