Identificação de GmERD 15, um novo fator de transcrição que controla a expressão do gene GmNRP-B em soja

• Ano de defesa: 2010
• Autor(a) principal: Alves, Murilo Siqueira
• Orientador(a): Fietto, Luciano Gomes
• Banca de defesa: Loureiro, Marcelo Ehlers , Zerbini Júnior, Francisco Murilo
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Bioquímica Agrícola
• Departamento: Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
• País: BR
• Palavras-chave em Português: GmERD 15; Soja
• Palavras-chave em Inglês: GmERD 15; Soybean
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
• Link de acesso: http://locus.ufv.br/handle/123456789/2420

Recently it was demonstrated through experiments using DNA microarrays that response pathways to endoplasmic reticulum and osmotic stresses converge with an increase in the expression of a group of coordinately regulated genes. Among these is GmNRP-B encodes an asparagine rich protein involved with events of programmed cell death. The main objectives of this work are the identification and characterization of transcription factors that control the expression of GmNRP-B. For these purposes the predict promoter of GmNRP-B was identified with bioinformatics tools and transcription fusions were built with the reporters genes HIS3 and LacZ. These constructs wereintegrated in the genome of Saccharomyces cerevisiae (strain W303), resulting in the transformants W303-pNRP-His/LacZ, that were used as hosts for transformation with a cDNA soybean library built in the vector pEXP-AD502.After library screenings 9 clones were selected and just one proved to be positive after a new transformation event. The isolated cDNA was found be highly similar to ERD15 from Arabidopsis and was named GmERD15. The deduced amino acid sequence showed that the protein possesses a conserved interaction domain with proteins that binds to poly A mRNA tails (PABP). The search for similar sequences in the Phytozome database showed that the gene is duplicated in the soybean genomeand belongs to a gene family represented by four additional copies in the soybean genome. Sequence comparison analyses of GmERD15 with plant orthologs showed that GmERD15 possesses a conserved PABP interaction domain in the amino-terminal portion and one divergent carboxi-terminal region. Based on clustering analysis of these proteins, they can be divided in at least 3 subfamilies and GmERD15 was grouped in a separated subfamily in relation to Arabidopsis protein. To confirm the interaction of GmERD15 with the predicted GmNRP-B promoter region we made deletions using the nucleotides -1000 upstream to the translation start codon leading to fragments with 700 and 350 bp. One-hybrid analysis in yeasts showed that this protein activated the expression of LacZ when this is under the control of GmNRP-B promoter with 700 and 1000 base pairs. However this activation decreased when the promoter was deleted to approximately 350 bp, indicating a possible loss of cis-elements. It was also verified that GmERD 15 possesses transactivation activity in yeasts. Furthermore, sequence analysis of GMERD15 showed the presence of a transcriptional activation domain in thecarboxi-terminal portion and the transient expression of GmERD 15 in soybean protoplasts led to an increase in GmNRP-B expression. GmERD 15 YFP protein fusions when expressed transiently in tobacco leaves were located in the citosol and in the nucleus. Collectively, these results indicate that GmERD 15 acts in the control of GmNRP-B gene expression revealing a new transfactor family in soybean.