Identificação de GmERD 15, um novo fator de transcrição que controla a expressão do gene GmNRP-B em soja
Autor(a) principal: | |
---|---|
Data de Publicação: | 2010 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | LOCUS Repositório Institucional da UFV |
Texto Completo: | http://locus.ufv.br/handle/123456789/2420 |
Resumo: | 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. |
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Alves, Murilo Siqueirahttp://lattes.cnpq.br/8216921216597311Fontes, Elizabeth Pacheco Batistahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781848H2Carvalho, Claudine Márciahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4794965T6Fietto, Luciano Gomeshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H8Loureiro, Marcelo Ehlershttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780851Y3Zerbini Júnior, Francisco Murilohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783743U52015-03-26T13:07:29Z2011-10-172015-03-26T13:07:29Z2010-02-12ALVES, Murilo Siqueira. Identification of the GmERD 15, a new transcription factor that controls the GmNRP-B expression in soybean. 2010. 80 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2010.http://locus.ufv.br/handle/123456789/2420Recently 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.Recentemente foi demonstrado através de experimentos de microarranjos de DNA que as vias de resposta aos estresses no retículo endoplasmático e osmótico convergem aumentando a expressão de um conjunto de genes coordenadamente regulados, dentre eles o gene GmNRP-B codifica uma proteína rica em asparagina e que está envolvida com eventos de morte celular programada em plantas. Este trabalho teve como objetivo principal a identificação e caracterização de fatores de transcrição que controlam a expressão de GmNRP-B, e que portanto constitui um importante componente da via integrativa dos estresses do retículo e osmótico. Para tanto a região promotora de GmNRP-B foi identificada em banco de dados e a partir dela foram construídas fusões de transcrição com os genes repórteres HIS3 e LacZ. Estas construções foram integradas no genoma de leveduras W303, resultando nos transformantes W303-pNRP-His/LacZ, que foram utilizadas como hospedeiras para transformação com uma biblioteca de cDNA de soja construída no vetor pEXP-AD502. Após a varredura da biblioteca, foram selecionados 9 clones, destes 9 apenas um manteve-se positivo após um novo evento de transformação. O sequenciamento do DNA do clone positivo e a comparação de sua seqüência com o banco de dados do Phytozome identificou um gene que codifica uma proteína similar a ERD15 de Arabidopsis. O gene de soja identificado foi denominado GmERD15 e a seqüência de aminoácidos deduzida mostrou que a proteína possui um domínio conservado de interação com proteínas que se ligam à cauda poli A de mRNAs (PABP). A busca por seqüências similares no banco dedados do Phytozome mostrou que o gene encontra-se duplicado no genoma da soja e que além dos dois genes outros 4 constituem esta família gênica em soja. A comparação da seqüência de aminoácidos deduzida com ortólogos ERD15 de diversos organismos mostrou que GmERD15 possui o domínio de interação com PABP conservado em sua porção amino-terminal e uma porção carboxi terminal divergente. A análise do agrupamento destas proteínas mostrou que podemos dividir esta família de proteínas em pelo menos 3 subfamílias sendo que GmERD15 encontra-se em uma subfamília separada da proteína de Arabidopsis. Para confirmar a interação da proteína GmERD15 com a região promotora de GmNRP-B foram realizadas deleções a partir do nucleotídeo -1000 do início de tradução originando fragmentos com 700 e 350 pares de bases. A análise destes promotores por monohíbrido em leveduras mostrou que esta proteína ativa fortemente a expressão de LacZ sob controle do promotor de GmNRP-B de 1000 e 700 pares de bases, porém esta ativação decai consideravelmente quando o promotor é deletado até aproximadamente 350 pares de bases, indicando uma grande perda de cis-elementos. Foi constatado também que GmERD 15 possui atividade de transativação em leveduras. Além disto a análise da sequência de aminoácidos mostrou a presença de um domínio de ativação transcricional na porção carboxi-terminal, e a expressão transiente de GmERD 15 em protoplastos de soja levou a um aumento da expressão de GmNRP-B. Quimeras de GmERD 15 com a proteína YFP expressas transientemente em folhas de tabaco localizam-se no citosol e no núcleo. Coletivamente, estes resultados indicam que GmERD 15 atua no controle da expressão do gene GmNRP-B revelando uma nova família de transfatores, que atuam nocontrole da expressão de genes em plantas.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de ViçosaMestrado em Bioquímica AgrícolaUFVBRBioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animalGmERD 15SojaGmERD 15SoybeanCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULARIdentificação de GmERD 15, um novo fator de transcrição que controla a expressão do gene GmNRP-B em sojaIdentification of the GmERD 15, a new transcription factor that controls the GmNRP-B expression in soybeaninfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf1304523https://locus.ufv.br//bitstream/123456789/2420/1/texto%20completo.pdf9e58f992543ea36131dfa73a9492c471MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain123807https://locus.ufv.br//bitstream/123456789/2420/2/texto%20completo.pdf.txt1c09742f7803ec6fe77ff17bedbba843MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3708https://locus.ufv.br//bitstream/123456789/2420/3/texto%20completo.pdf.jpga196c149e2157a0b86718fc8f56dc101MD53123456789/24202016-04-08 23:02:20.144oai:locus.ufv.br:123456789/2420Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-09T02:02:20LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
Identificação de GmERD 15, um novo fator de transcrição que controla a expressão do gene GmNRP-B em soja |
dc.title.alternative.eng.fl_str_mv |
Identification of the GmERD 15, a new transcription factor that controls the GmNRP-B expression in soybean |
title |
Identificação de GmERD 15, um novo fator de transcrição que controla a expressão do gene GmNRP-B em soja |
spellingShingle |
Identificação de GmERD 15, um novo fator de transcrição que controla a expressão do gene GmNRP-B em soja Alves, Murilo Siqueira GmERD 15 Soja GmERD 15 Soybean CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
title_short |
Identificação de GmERD 15, um novo fator de transcrição que controla a expressão do gene GmNRP-B em soja |
title_full |
Identificação de GmERD 15, um novo fator de transcrição que controla a expressão do gene GmNRP-B em soja |
title_fullStr |
Identificação de GmERD 15, um novo fator de transcrição que controla a expressão do gene GmNRP-B em soja |
title_full_unstemmed |
Identificação de GmERD 15, um novo fator de transcrição que controla a expressão do gene GmNRP-B em soja |
title_sort |
Identificação de GmERD 15, um novo fator de transcrição que controla a expressão do gene GmNRP-B em soja |
author |
Alves, Murilo Siqueira |
author_facet |
Alves, Murilo Siqueira |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/8216921216597311 |
dc.contributor.author.fl_str_mv |
Alves, Murilo Siqueira |
dc.contributor.advisor-co1.fl_str_mv |
Fontes, Elizabeth Pacheco Batista |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781848H2 |
dc.contributor.advisor-co2.fl_str_mv |
Carvalho, Claudine Márcia |
dc.contributor.advisor-co2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4794965T6 |
dc.contributor.advisor1.fl_str_mv |
Fietto, Luciano Gomes |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H8 |
dc.contributor.referee1.fl_str_mv |
Loureiro, Marcelo Ehlers |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780851Y3 |
dc.contributor.referee2.fl_str_mv |
Zerbini Júnior, Francisco Murilo |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783743U5 |
contributor_str_mv |
Fontes, Elizabeth Pacheco Batista Carvalho, Claudine Márcia Fietto, Luciano Gomes Loureiro, Marcelo Ehlers Zerbini Júnior, Francisco Murilo |
dc.subject.por.fl_str_mv |
GmERD 15 Soja |
topic |
GmERD 15 Soja GmERD 15 Soybean CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
dc.subject.eng.fl_str_mv |
GmERD 15 Soybean |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
description |
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. |
publishDate |
2010 |
dc.date.issued.fl_str_mv |
2010-02-12 |
dc.date.available.fl_str_mv |
2011-10-17 2015-03-26T13:07:29Z |
dc.date.accessioned.fl_str_mv |
2015-03-26T13:07:29Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
ALVES, Murilo Siqueira. Identification of the GmERD 15, a new transcription factor that controls the GmNRP-B expression in soybean. 2010. 80 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2010. |
dc.identifier.uri.fl_str_mv |
http://locus.ufv.br/handle/123456789/2420 |
identifier_str_mv |
ALVES, Murilo Siqueira. Identification of the GmERD 15, a new transcription factor that controls the GmNRP-B expression in soybean. 2010. 80 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2010. |
url |
http://locus.ufv.br/handle/123456789/2420 |
dc.language.iso.fl_str_mv |
por |
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por |
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info:eu-repo/semantics/openAccess |
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Universidade Federal de Viçosa |
dc.publisher.program.fl_str_mv |
Mestrado em Bioquímica Agrícola |
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UFV |
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BR |
dc.publisher.department.fl_str_mv |
Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal |
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Universidade Federal de Viçosa |
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reponame:LOCUS Repositório Institucional da UFV instname:Universidade Federal de Viçosa (UFV) instacron:UFV |
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