Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris
Autor(a) principal: | |
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Data de Publicação: | 2016 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da UFG |
Texto Completo: | http://repositorio.bc.ufg.br/tede/handle/tede/7365 |
Resumo: | In the last decades the search for new renewable energy sources have increased and one of the emerged technologies was the production of ethanol from lignocellulosic biomass (second generation ethanol). For obtaining fermentable sugars from biomass, a complex of enzymes and proteins that acts on polysaccharides of the plant cell wall is required. Expansins are proteins that can help enzymes in the degradation process, promoting the loosening of the cell wall of plants by breaking hydrogen bonds between cellulose fibers and hemicellulose. The superfamily of expansins has been described mainly in plants, although they are also found in fungi and bacteria. Four families are described to this superfamily: α-expansins, β-expansins, expansins-like A and expansins-like B. The α-expansins are present in most eudicots, whereas the β-expansins are involved in cellular expansion process of the monocots family Poaceae. To analyze the sequence and to verify the functional activity of a β-expansin on filter paper, in this work a gene of β-expansin (expb11) of sugarcane was isolated, cloned and expressed in the yeast Pichia pastoris. Nucleotide sequences of expansin genes from sugarcane were initially obtained from the public SUCEST database using reference sequences of expansins from sorghum, maize and rice as query. A total of 227 ESTs were identified. These ESTs were submitted to clustering and 30 contigs and 92 singletons were obtained. Specific oligos were designed and used to capture the genomic fragment corresponding to the expb11 gene by PCR. The coding DNA fragment of expb11 was obtained by RT-PCR from the total RNA extracted from stalks of the RB867515 sugarcane cultivar. This fragment was cloned into the pGEM-T Easy vector and subcloned into the pPIC9 vector. Genomic and cDNA fragments were sequenced. The expression cassette generated by subcloning into the pPIC9 expression vector was integrated into the P. pastoris strain GS115 genome, but it has not been possible to detect a functional activity of expansin. The expb11 gene comprises 1367 bp, containing 3 introns, and its cDNA is 801 bp long, with an ORF of 776 bp. The alignment of sugarcane and sorghum sequences of expb11 showed that the two species share a 95% sequence identity along these genes and that the gene structure is highly conserved in these species. The in silico analysis of the putative EXPB11 amino acid sequence allowed the detection of two conserved domains, one similar to the GH45 family, and the other to the carbohydrate binding module (CBM). The putative EXPB11 protein has a predicted molecular weight of approximately 26.4 kDa, an isoelectric point (pI) of 4.88, and contains two glycosylation sites. Our results confirmed that the isolated and cloned expb11 gene corresponds to a β-expansin gene from sugarcane, paving the way to the expression, isolation and use of recombinant EXPB11 in the mix of enzymes and proteins for lignocellulosic biomass degradation. |
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Coelho, Alexandre Siqueira Guedeshttp://lattes.cnpq.br/0840926305216925Faria, Fabrícia Paula dehttp://lattes.cnpq.br/3739169267521003Coelho, Alexandre Siqueira GuedesSilva, Silvana PetrofezaGeorg, Raphaela de CastroSiqueira, Saulo José LinharesColussi, Francielihttp://lattes.cnpq.br/8293395800807282Costa, Ilítia Ganaê de Oliveira2017-05-26T13:43:28Z2016-08-31COSTA, I. G. O. Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris. 2016. 95 f. Dissertação (Mestrado em Genética e Melhoramento de Plantas) - Universidade Federal de Goiás, Goiânia, 2016.http://repositorio.bc.ufg.br/tede/handle/tede/7365In the last decades the search for new renewable energy sources have increased and one of the emerged technologies was the production of ethanol from lignocellulosic biomass (second generation ethanol). For obtaining fermentable sugars from biomass, a complex of enzymes and proteins that acts on polysaccharides of the plant cell wall is required. Expansins are proteins that can help enzymes in the degradation process, promoting the loosening of the cell wall of plants by breaking hydrogen bonds between cellulose fibers and hemicellulose. The superfamily of expansins has been described mainly in plants, although they are also found in fungi and bacteria. Four families are described to this superfamily: α-expansins, β-expansins, expansins-like A and expansins-like B. The α-expansins are present in most eudicots, whereas the β-expansins are involved in cellular expansion process of the monocots family Poaceae. To analyze the sequence and to verify the functional activity of a β-expansin on filter paper, in this work a gene of β-expansin (expb11) of sugarcane was isolated, cloned and expressed in the yeast Pichia pastoris. Nucleotide sequences of expansin genes from sugarcane were initially obtained from the public SUCEST database using reference sequences of expansins from sorghum, maize and rice as query. A total of 227 ESTs were identified. These ESTs were submitted to clustering and 30 contigs and 92 singletons were obtained. Specific oligos were designed and used to capture the genomic fragment corresponding to the expb11 gene by PCR. The coding DNA fragment of expb11 was obtained by RT-PCR from the total RNA extracted from stalks of the RB867515 sugarcane cultivar. This fragment was cloned into the pGEM-T Easy vector and subcloned into the pPIC9 vector. Genomic and cDNA fragments were sequenced. The expression cassette generated by subcloning into the pPIC9 expression vector was integrated into the P. pastoris strain GS115 genome, but it has not been possible to detect a functional activity of expansin. The expb11 gene comprises 1367 bp, containing 3 introns, and its cDNA is 801 bp long, with an ORF of 776 bp. The alignment of sugarcane and sorghum sequences of expb11 showed that the two species share a 95% sequence identity along these genes and that the gene structure is highly conserved in these species. The in silico analysis of the putative EXPB11 amino acid sequence allowed the detection of two conserved domains, one similar to the GH45 family, and the other to the carbohydrate binding module (CBM). The putative EXPB11 protein has a predicted molecular weight of approximately 26.4 kDa, an isoelectric point (pI) of 4.88, and contains two glycosylation sites. Our results confirmed that the isolated and cloned expb11 gene corresponds to a β-expansin gene from sugarcane, paving the way to the expression, isolation and use of recombinant EXPB11 in the mix of enzymes and proteins for lignocellulosic biomass degradation.Nos últimos anos tem crescido a busca por novas fontes de energia renováveis, e uma das tecnologias utilizadas é a produção de etanol a partir da biomassa lignocelulósica (etanol de segunda geração). Para a obtenção de açúcares fermentáveis a partir dessa biomassa, é necessário um complexo de enzimas e proteínas que atuarão sobre os polissacarídeos da parede celular vegetal. As expansinas são proteínas que podem auxiliar as enzimas no processo de degradação, promovendo o afrouxamento da parede celular das plantas pelo rompimento de ligações de hidrogênio entre fibras de celulose e hemicelulose. A superfamília das expansinas foi descrita principalmente em plantas, entretanto, há relatos em fungos e bactérias. São descritas 4 famílias de expansinas: α-expansinas, β-expansinas, expansinas like-A e expansinas like-B. As α e β-expansinas já são caracterizadas, desempenhando importante função no crescimento vegetal. As α-expansinas são mais presentes em plantas dicotiledôneas, enquanto que as β-expansinas estão envolvidas no processo de expansão celular de monocotiledôneas da família Poaceae. Para analisar a sequência e verificar a atividade funcional de uma β-expansina sobre bagaço de cana-de-açúcar, neste trabalho foi isolado, clonado e expresso um gene de β-expansina (expb11) de cana-de-açúcar na levedura Pichia pastoris. A sequência de nucleotídeos do gene de β-expansina da cana-de-açúcar foi obtida a partir da biblioteca de cDNA de cana-de-açúcar do projeto SUCEST. Sequências de referências de expansinas de sorgo, milho e arroz foram utilizadas para identificar ESTs de expansinas no banco de dados SUCEST. Foram identificadas 227 ESTs. Essas ESTs foram submetidas à clusterização e foram obtidos 30 contigs e 92 singletons. Após o desenho de oligonucleotídeos, o fragmento genômico correspondente ao gene expb11 foi amplificado por PCR. A região codante do gene expb11 foi obtida por RT-PCR a partir de RNA total obtido de colmo da cultivar RB867515. Esse material foi clonado no vetor pGEM-T Easy e subclonado no vetor pPIC9. Os fragmentos genômico e cDNA obtidos foram analisados por sequenciamento. O cassete de expressão gerado pela subclonagem em vetor de expressão pPIC9 foi integrado no genoma de P. pastoris linhagem GS115, mas não foi possível detectar atividade funcional da expansina. O gene expb11 possui 1367 pb, contendo 3 íntrons, enquanto o cDNA 801 pb. Pelo alinhamento das sequências genômicas do gene expb11 obtidas de cana-de-açúcar e de sorgo, foi possível se observar que estes genes possuem 95 % de identidade, e estrutura de íntrons/éxons semelhantes. Pela análise in silico da sequência de aminoácidos da proteína EXPB11 foi possível verificar que ela possui dois domínios, um similar ao da família GH45, e outro similar ao módulo de ligação ao carboidrato (CBM). A proteína EXPB11 possui peso molecular de aproximadamente 26,4 kDa, pI 4,88, e contém dois sítios de glicosilação. Os resultados obtidos através das análises in silico mostraram que o gene expb11, isolado e clonado, corresponde a um gene de β-expansina de cana-de-açúcar, e ao ser produzida, a EXPB11 recombinante poderá integrar o mix de enzimas e proteínas para a degradação de biomassa lignocelulósica.Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-26T13:43:05Z No. of bitstreams: 2 Dissertação - Ilítia Ganaê de Oliveira Costa - 2016.pdf: 5422422 bytes, checksum: da6f13721ce50902b9fe7d9ac3713ab5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-26T13:43:28Z (GMT) No. of bitstreams: 2 Dissertação - Ilítia Ganaê de Oliveira Costa - 2016.pdf: 5422422 bytes, checksum: da6f13721ce50902b9fe7d9ac3713ab5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2017-05-26T13:43:28Z (GMT). No. of bitstreams: 2 Dissertação - Ilítia Ganaê de Oliveira Costa - 2016.pdf: 5422422 bytes, checksum: da6f13721ce50902b9fe7d9ac3713ab5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-08-31Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESapplication/pdfporUniversidade Federal de GoiásPrograma de Pós-graduação em Genética e Melhoramento de Plantas (EAEA)UFGBrasilEscola de Agronomia e Engenharia de Alimentos - EAEA (RG)http://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessβ-expansinaSaccharum officinarumexpressão heterólogaPichia pastorisβ-expansinSaccharum officinarumHeterologous expressionPichia pastorisGENETICA::GENETICA VEGETALClonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastorisCloning and expression of a β-expansin of sugarcane in the yeast Pichia pastorisinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis-33250994043618731196006006006004500684695727928426-73979202484192807162075167498588264571reponame:Biblioteca Digital de Teses e Dissertações da UFGinstname:Universidade Federal de Goiás (UFG)instacron:UFGLICENSElicense.txtlicense.txttext/plain; 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dc.title.eng.fl_str_mv |
Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris |
dc.title.alternative.eng.fl_str_mv |
Cloning and expression of a β-expansin of sugarcane in the yeast Pichia pastoris |
title |
Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris |
spellingShingle |
Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris Costa, Ilítia Ganaê de Oliveira β-expansina Saccharum officinarum expressão heteróloga Pichia pastoris β-expansin Saccharum officinarum Heterologous expression Pichia pastoris GENETICA::GENETICA VEGETAL |
title_short |
Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris |
title_full |
Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris |
title_fullStr |
Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris |
title_full_unstemmed |
Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris |
title_sort |
Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris |
author |
Costa, Ilítia Ganaê de Oliveira |
author_facet |
Costa, Ilítia Ganaê de Oliveira |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Coelho, Alexandre Siqueira Guedes |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/0840926305216925 |
dc.contributor.advisor-co1.fl_str_mv |
Faria, Fabrícia Paula de |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/3739169267521003 |
dc.contributor.referee1.fl_str_mv |
Coelho, Alexandre Siqueira Guedes |
dc.contributor.referee2.fl_str_mv |
Silva, Silvana Petrofeza |
dc.contributor.referee3.fl_str_mv |
Georg, Raphaela de Castro |
dc.contributor.referee4.fl_str_mv |
Siqueira, Saulo José Linhares |
dc.contributor.referee5.fl_str_mv |
Colussi, Francieli |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/8293395800807282 |
dc.contributor.author.fl_str_mv |
Costa, Ilítia Ganaê de Oliveira |
contributor_str_mv |
Coelho, Alexandre Siqueira Guedes Faria, Fabrícia Paula de Coelho, Alexandre Siqueira Guedes Silva, Silvana Petrofeza Georg, Raphaela de Castro Siqueira, Saulo José Linhares Colussi, Francieli |
dc.subject.por.fl_str_mv |
β-expansina Saccharum officinarum expressão heteróloga Pichia pastoris |
topic |
β-expansina Saccharum officinarum expressão heteróloga Pichia pastoris β-expansin Saccharum officinarum Heterologous expression Pichia pastoris GENETICA::GENETICA VEGETAL |
dc.subject.eng.fl_str_mv |
β-expansin Saccharum officinarum Heterologous expression Pichia pastoris |
dc.subject.cnpq.fl_str_mv |
GENETICA::GENETICA VEGETAL |
description |
In the last decades the search for new renewable energy sources have increased and one of the emerged technologies was the production of ethanol from lignocellulosic biomass (second generation ethanol). For obtaining fermentable sugars from biomass, a complex of enzymes and proteins that acts on polysaccharides of the plant cell wall is required. Expansins are proteins that can help enzymes in the degradation process, promoting the loosening of the cell wall of plants by breaking hydrogen bonds between cellulose fibers and hemicellulose. The superfamily of expansins has been described mainly in plants, although they are also found in fungi and bacteria. Four families are described to this superfamily: α-expansins, β-expansins, expansins-like A and expansins-like B. The α-expansins are present in most eudicots, whereas the β-expansins are involved in cellular expansion process of the monocots family Poaceae. To analyze the sequence and to verify the functional activity of a β-expansin on filter paper, in this work a gene of β-expansin (expb11) of sugarcane was isolated, cloned and expressed in the yeast Pichia pastoris. Nucleotide sequences of expansin genes from sugarcane were initially obtained from the public SUCEST database using reference sequences of expansins from sorghum, maize and rice as query. A total of 227 ESTs were identified. These ESTs were submitted to clustering and 30 contigs and 92 singletons were obtained. Specific oligos were designed and used to capture the genomic fragment corresponding to the expb11 gene by PCR. The coding DNA fragment of expb11 was obtained by RT-PCR from the total RNA extracted from stalks of the RB867515 sugarcane cultivar. This fragment was cloned into the pGEM-T Easy vector and subcloned into the pPIC9 vector. Genomic and cDNA fragments were sequenced. The expression cassette generated by subcloning into the pPIC9 expression vector was integrated into the P. pastoris strain GS115 genome, but it has not been possible to detect a functional activity of expansin. The expb11 gene comprises 1367 bp, containing 3 introns, and its cDNA is 801 bp long, with an ORF of 776 bp. The alignment of sugarcane and sorghum sequences of expb11 showed that the two species share a 95% sequence identity along these genes and that the gene structure is highly conserved in these species. The in silico analysis of the putative EXPB11 amino acid sequence allowed the detection of two conserved domains, one similar to the GH45 family, and the other to the carbohydrate binding module (CBM). The putative EXPB11 protein has a predicted molecular weight of approximately 26.4 kDa, an isoelectric point (pI) of 4.88, and contains two glycosylation sites. Our results confirmed that the isolated and cloned expb11 gene corresponds to a β-expansin gene from sugarcane, paving the way to the expression, isolation and use of recombinant EXPB11 in the mix of enzymes and proteins for lignocellulosic biomass degradation. |
publishDate |
2016 |
dc.date.issued.fl_str_mv |
2016-08-31 |
dc.date.accessioned.fl_str_mv |
2017-05-26T13:43:28Z |
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 |
COSTA, I. G. O. Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris. 2016. 95 f. Dissertação (Mestrado em Genética e Melhoramento de Plantas) - Universidade Federal de Goiás, Goiânia, 2016. |
dc.identifier.uri.fl_str_mv |
http://repositorio.bc.ufg.br/tede/handle/tede/7365 |
identifier_str_mv |
COSTA, I. G. O. Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris. 2016. 95 f. Dissertação (Mestrado em Genética e Melhoramento de Plantas) - Universidade Federal de Goiás, Goiânia, 2016. |
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http://repositorio.bc.ufg.br/tede/handle/tede/7365 |
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por |
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por |
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2075167498588264571 |
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http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
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http://creativecommons.org/licenses/by-nc-nd/4.0/ |
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openAccess |
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Universidade Federal de Goiás |
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Programa de Pós-graduação em Genética e Melhoramento de Plantas (EAEA) |
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UFG |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
Escola de Agronomia e Engenharia de Alimentos - EAEA (RG) |
publisher.none.fl_str_mv |
Universidade Federal de Goiás |
dc.source.none.fl_str_mv |
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Biblioteca Digital de Teses e Dissertações da UFG |
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