Transformação genética de nós cotiledonares de soja visando à co-supressão do gene da lisofosfatidilcolina aciltransferase

Detalhes bibliográficos
Autor(a) principal: Martins, Polyana Kelly
Data de Publicação: 2007
Tipo de documento: Tese
Idioma: por
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://locus.ufv.br/handle/123456789/1417
Resumo: The soybean (Glycine max L. Merrill) is one of the most important crops of the world and the biggest in Brazil in cultivated area. Brazil is the second largest world producer of these grain legumes, producing 56.3 million tons in the harvest year 2006/2007 (www.conab.gov.br). Modifications using techniques of genetic engineering can facilitate the fast development of new varieties with agronomic and industrial characteristics of interest. Alterations in the relative proportions of the fatty acids may be reached through genetic transformation of plants by gene silencing. Among these enzymes are desaturases, phosphotransferases and acyltransferases, involved in the fatty acids biosynthesis. The objectives of this work were to isolate the fragment of the soybean lysophosphatidycholine acyltransferase gene (LPCAT), to construct an expression cassette containing this fragment driven by the gene promoter of the α subunit of soybean storage protein β - conglycinine (seed-specific promoter) and to transform soybean cotyledonary node with this construction, seeking to the co-suppression of the LPCAT gene. The RT-PCR analysis showed that the LPCAT gene is expressed in all of the stages of seed development. The cassette of approximately 800 pb containing the seed-specific promoter sequence and of part of the gene LPCAT was cloned in the binary vector pCAMBIA 1304, whose T-DNA presents genes for resistance to the antibiotics kanamicina, for selection in bacteria, and Hygromycin B for selection in plants. The cloning was confirmed through PCR, enzymatic restriction and sequencing. The construction cloned in the vector pCAMBIA 1304 was used for soybean transformation by sonication using Agrobacterium tumefaciens. Mature soybean seeds were sterilized and germinated in B5 medium during, approximately, five days. From a single seedling, two explants were obtained, that were removed the root, followed by most of the hypocotyl and finally separated into two cotyledons. These explants were transformed with bacterial suspension by sonication and acetosyringone addition. After three days of co-cultivation, the cotyledonary node were then transferred to shoot induction medium (SIM) without selective agent, for 14 days. Soon afterwards, it was added to the medium SIM 5 mg/L of Hygromycin B for selection of transformed shoots. The selected explants were transferred to a shoot elongation medium (SEM), still with Hygromycin B, and later for the rooting medium (RM) without the selective agent. Two months after inoculation with A. tumefaciens, multiple shoots were produced in the node area resistant to Hygromycin B that were in direct contact with the medium. Leaf DNA analyses from plants in phase of acclimatization allowed the detection of several transformation events. Seventeen transgenic plants were confirmed by PCR analyses with specific primers for sequences present only in transformed plants.
id UFV_5038d0170ffa64763265a2cebca1a0a5
oai_identifier_str oai:locus.ufv.br:123456789/1417
network_acronym_str UFV
network_name_str LOCUS Repositório Institucional da UFV
repository_id_str 2145
spelling Martins, Polyana Kellyhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4770827E6Barros, Everaldo Gonçalves dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781285J6Otoni, Wagner Camposhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4786133Y6Moreira, Maurílio Alveshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4796105P2Lima, Andréia Barcelos Passoshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4766019H4Marcelino, Francismar Corrêahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4706159Y32015-03-26T12:45:46Z2007-07-242015-03-26T12:45:46Z2007-04-02MARTINS, Polyana Kelly. Genetic transformation of soybean cotyledonary node seeking the cosuppression of the lysophosphatidycholine acyltransferase. 2007. 94 f. Tese (Doutorado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2007.http://locus.ufv.br/handle/123456789/1417The soybean (Glycine max L. Merrill) is one of the most important crops of the world and the biggest in Brazil in cultivated area. Brazil is the second largest world producer of these grain legumes, producing 56.3 million tons in the harvest year 2006/2007 (www.conab.gov.br). Modifications using techniques of genetic engineering can facilitate the fast development of new varieties with agronomic and industrial characteristics of interest. Alterations in the relative proportions of the fatty acids may be reached through genetic transformation of plants by gene silencing. Among these enzymes are desaturases, phosphotransferases and acyltransferases, involved in the fatty acids biosynthesis. The objectives of this work were to isolate the fragment of the soybean lysophosphatidycholine acyltransferase gene (LPCAT), to construct an expression cassette containing this fragment driven by the gene promoter of the α subunit of soybean storage protein β - conglycinine (seed-specific promoter) and to transform soybean cotyledonary node with this construction, seeking to the co-suppression of the LPCAT gene. The RT-PCR analysis showed that the LPCAT gene is expressed in all of the stages of seed development. The cassette of approximately 800 pb containing the seed-specific promoter sequence and of part of the gene LPCAT was cloned in the binary vector pCAMBIA 1304, whose T-DNA presents genes for resistance to the antibiotics kanamicina, for selection in bacteria, and Hygromycin B for selection in plants. The cloning was confirmed through PCR, enzymatic restriction and sequencing. The construction cloned in the vector pCAMBIA 1304 was used for soybean transformation by sonication using Agrobacterium tumefaciens. Mature soybean seeds were sterilized and germinated in B5 medium during, approximately, five days. From a single seedling, two explants were obtained, that were removed the root, followed by most of the hypocotyl and finally separated into two cotyledons. These explants were transformed with bacterial suspension by sonication and acetosyringone addition. After three days of co-cultivation, the cotyledonary node were then transferred to shoot induction medium (SIM) without selective agent, for 14 days. Soon afterwards, it was added to the medium SIM 5 mg/L of Hygromycin B for selection of transformed shoots. The selected explants were transferred to a shoot elongation medium (SEM), still with Hygromycin B, and later for the rooting medium (RM) without the selective agent. Two months after inoculation with A. tumefaciens, multiple shoots were produced in the node area resistant to Hygromycin B that were in direct contact with the medium. Leaf DNA analyses from plants in phase of acclimatization allowed the detection of several transformation events. Seventeen transgenic plants were confirmed by PCR analyses with specific primers for sequences present only in transformed plants.A soja (Glycine max L. Merrill) é uma das mais importantes culturas do mundo e a maior do Brasil em área plantada. O Brasil é o segundo maior produtor mundial desta leguminosa atingindo 56,3 milhões de toneladas na safra 2006/2007. (www.conab.gov.br). Assim, modificações usando técnicas de engenharia genética podem facilitar o rápido desenvolvimento de novas variedades com características de interesse agronômico e industrial. Alterações nas proporções relativas dos ácidos graxos na fração óleo podem ser alcançadas por meio do silenciamento gênico de enzimas do tipo aciltransferases, fosfotransferases e dessaturases, envolvidas na biossíntese de ácidos graxos. Os objetivos deste trabalho foram: isolar um fragmento do gene da lisofosfatidilcolina aciltransferase (LPCAT) de soja, construir um cassete de expressão, contendo este fragmento, sob o controle da região promotora do gene da subunidade α da β-conglicinina (promotor semente-específico) e transformar nós cotiledonares de soja com esta construção, visando à co-supressão do gene LPCAT. A análise por RT-PCR mostrou que o gene LPCAT é expresso em todos os estádios de desenvolvimento da semente. O cassete de aproximadamente 800 pb contendo a seqüência do promotor semente-específico e de parte do gene LPCAT foi clonado no vetor binário pCAMBIA 1304, cujo T-DNA apresenta genes para resistência aos antibióticos canamicina, para seleção em bactérias, e Higromicina B para seleção em plantas. A clonagem foi confirmada por meio de PCR, restrição enzimática e seqüenciamento. A construção clonada no vetor pCAMBIA 1304 foi utilizada para a transformação de soja via Agrobacterium tumefaciens e sonicação. Sementes maduras de soja foram desinfetadas e germinadas em meio B5 durante, aproximadamente, 5 dias. A partir de uma semente, dois explantes foram obtidos, primeiramente removendo-se a raiz, em seguida, a maior parte do hipocótilo e finalmente um corte vertical feito para separar os dois cotilédones. Estes explantes foram transformados com suspensão bacteriana mediante sonicação e adição de acetoseringona. Após 3 dias de co-cultivo, os nós-cotiledonares foram transferidos para meio de indução (SIM) sem agente seletivo, durante 14 dias. Em seguida, foi adicionado ao meio SIM 5mg/L de Higromicina B para seleção dos transformantes. Os explantes que sobreviveram a essa seleção foram transferidos para o meio de elongação (SEM), ainda com Higromicina B, e depois para o meio de enraizamento (RM) sem o agente seletivo. Dois meses após inoculação com A. tumefaciens, múltiplos brotos foram produzidos nas regiões nodais resistentes a Higromicina B que estavam em contato direto com o meio. Análises a partir do DNA de folhas de plantas em fase de aclimatação permitiram a detecção de vários eventos de transformação. Dezessete eventos foram confirmados por reações de PCR com primers específicos para seqüências presentes apenas em plantas transformadas.Conselho Nacional de Desenvolvimento Científico e Tecnológicoapplication/pdfporUniversidade Federal de ViçosaDoutorado em Genética e MelhoramentoUFVBRGenética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; MeÁcidos graxosBiossínteseTransformação genética vegetalAgrobacterium tumefaciensFatty acidsBiosynthesisGenetic transformation of plantsAgrobacterium tumefaciensCNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOSTransformação genética de nós cotiledonares de soja visando à co-supressão do gene da lisofosfatidilcolina aciltransferaseGenetic transformation of soybean cotyledonary node seeking the cosuppression of the lysophosphatidycholine acyltransferaseinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf920630https://locus.ufv.br//bitstream/123456789/1417/1/texto%20completo.pdf1cb09b3dc99fe9425960ca39095e2663MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain175173https://locus.ufv.br//bitstream/123456789/1417/2/texto%20completo.pdf.txt5f0f1eb8aa135858f721c08dd70c4337MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3647https://locus.ufv.br//bitstream/123456789/1417/3/texto%20completo.pdf.jpgac145e50fb72528f71ea53f6b849c023MD53123456789/14172016-04-07 23:08:07.041oai:locus.ufv.br:123456789/1417Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-08T02:08:07LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.por.fl_str_mv Transformação genética de nós cotiledonares de soja visando à co-supressão do gene da lisofosfatidilcolina aciltransferase
dc.title.alternative.eng.fl_str_mv Genetic transformation of soybean cotyledonary node seeking the cosuppression of the lysophosphatidycholine acyltransferase
title Transformação genética de nós cotiledonares de soja visando à co-supressão do gene da lisofosfatidilcolina aciltransferase
spellingShingle Transformação genética de nós cotiledonares de soja visando à co-supressão do gene da lisofosfatidilcolina aciltransferase
Martins, Polyana Kelly
Ácidos graxos
Biossíntese
Transformação genética vegetal
Agrobacterium tumefaciens
Fatty acids
Biosynthesis
Genetic transformation of plants
Agrobacterium tumefaciens
CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS
title_short Transformação genética de nós cotiledonares de soja visando à co-supressão do gene da lisofosfatidilcolina aciltransferase
title_full Transformação genética de nós cotiledonares de soja visando à co-supressão do gene da lisofosfatidilcolina aciltransferase
title_fullStr Transformação genética de nós cotiledonares de soja visando à co-supressão do gene da lisofosfatidilcolina aciltransferase
title_full_unstemmed Transformação genética de nós cotiledonares de soja visando à co-supressão do gene da lisofosfatidilcolina aciltransferase
title_sort Transformação genética de nós cotiledonares de soja visando à co-supressão do gene da lisofosfatidilcolina aciltransferase
author Martins, Polyana Kelly
author_facet Martins, Polyana Kelly
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4770827E6
dc.contributor.author.fl_str_mv Martins, Polyana Kelly
dc.contributor.advisor-co1.fl_str_mv Barros, Everaldo Gonçalves de
dc.contributor.advisor-co1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781285J6
dc.contributor.advisor-co2.fl_str_mv Otoni, Wagner Campos
dc.contributor.advisor-co2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4786133Y6
dc.contributor.advisor1.fl_str_mv Moreira, Maurílio Alves
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4796105P2
dc.contributor.referee1.fl_str_mv Lima, Andréia Barcelos Passos
dc.contributor.referee1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4766019H4
dc.contributor.referee2.fl_str_mv Marcelino, Francismar Corrêa
dc.contributor.referee2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4706159Y3
contributor_str_mv Barros, Everaldo Gonçalves de
Otoni, Wagner Campos
Moreira, Maurílio Alves
Lima, Andréia Barcelos Passos
Marcelino, Francismar Corrêa
dc.subject.por.fl_str_mv Ácidos graxos
Biossíntese
Transformação genética vegetal
Agrobacterium tumefaciens
topic Ácidos graxos
Biossíntese
Transformação genética vegetal
Agrobacterium tumefaciens
Fatty acids
Biosynthesis
Genetic transformation of plants
Agrobacterium tumefaciens
CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS
dc.subject.eng.fl_str_mv Fatty acids
Biosynthesis
Genetic transformation of plants
Agrobacterium tumefaciens
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS
description The soybean (Glycine max L. Merrill) is one of the most important crops of the world and the biggest in Brazil in cultivated area. Brazil is the second largest world producer of these grain legumes, producing 56.3 million tons in the harvest year 2006/2007 (www.conab.gov.br). Modifications using techniques of genetic engineering can facilitate the fast development of new varieties with agronomic and industrial characteristics of interest. Alterations in the relative proportions of the fatty acids may be reached through genetic transformation of plants by gene silencing. Among these enzymes are desaturases, phosphotransferases and acyltransferases, involved in the fatty acids biosynthesis. The objectives of this work were to isolate the fragment of the soybean lysophosphatidycholine acyltransferase gene (LPCAT), to construct an expression cassette containing this fragment driven by the gene promoter of the α subunit of soybean storage protein β - conglycinine (seed-specific promoter) and to transform soybean cotyledonary node with this construction, seeking to the co-suppression of the LPCAT gene. The RT-PCR analysis showed that the LPCAT gene is expressed in all of the stages of seed development. The cassette of approximately 800 pb containing the seed-specific promoter sequence and of part of the gene LPCAT was cloned in the binary vector pCAMBIA 1304, whose T-DNA presents genes for resistance to the antibiotics kanamicina, for selection in bacteria, and Hygromycin B for selection in plants. The cloning was confirmed through PCR, enzymatic restriction and sequencing. The construction cloned in the vector pCAMBIA 1304 was used for soybean transformation by sonication using Agrobacterium tumefaciens. Mature soybean seeds were sterilized and germinated in B5 medium during, approximately, five days. From a single seedling, two explants were obtained, that were removed the root, followed by most of the hypocotyl and finally separated into two cotyledons. These explants were transformed with bacterial suspension by sonication and acetosyringone addition. After three days of co-cultivation, the cotyledonary node were then transferred to shoot induction medium (SIM) without selective agent, for 14 days. Soon afterwards, it was added to the medium SIM 5 mg/L of Hygromycin B for selection of transformed shoots. The selected explants were transferred to a shoot elongation medium (SEM), still with Hygromycin B, and later for the rooting medium (RM) without the selective agent. Two months after inoculation with A. tumefaciens, multiple shoots were produced in the node area resistant to Hygromycin B that were in direct contact with the medium. Leaf DNA analyses from plants in phase of acclimatization allowed the detection of several transformation events. Seventeen transgenic plants were confirmed by PCR analyses with specific primers for sequences present only in transformed plants.
publishDate 2007
dc.date.available.fl_str_mv 2007-07-24
2015-03-26T12:45:46Z
dc.date.issued.fl_str_mv 2007-04-02
dc.date.accessioned.fl_str_mv 2015-03-26T12:45:46Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/doctoralThesis
format doctoralThesis
status_str publishedVersion
dc.identifier.citation.fl_str_mv MARTINS, Polyana Kelly. Genetic transformation of soybean cotyledonary node seeking the cosuppression of the lysophosphatidycholine acyltransferase. 2007. 94 f. Tese (Doutorado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2007.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/1417
identifier_str_mv MARTINS, Polyana Kelly. Genetic transformation of soybean cotyledonary node seeking the cosuppression of the lysophosphatidycholine acyltransferase. 2007. 94 f. Tese (Doutorado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2007.
url http://locus.ufv.br/handle/123456789/1417
dc.language.iso.fl_str_mv por
language por
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Universidade Federal de Viçosa
dc.publisher.program.fl_str_mv Doutorado em Genética e Melhoramento
dc.publisher.initials.fl_str_mv UFV
dc.publisher.country.fl_str_mv BR
dc.publisher.department.fl_str_mv Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me
publisher.none.fl_str_mv Universidade Federal de Viçosa
dc.source.none.fl_str_mv reponame:LOCUS Repositório Institucional da UFV
instname:Universidade Federal de Viçosa (UFV)
instacron:UFV
instname_str Universidade Federal de Viçosa (UFV)
instacron_str UFV
institution UFV
reponame_str LOCUS Repositório Institucional da UFV
collection LOCUS Repositório Institucional da UFV
bitstream.url.fl_str_mv https://locus.ufv.br//bitstream/123456789/1417/1/texto%20completo.pdf
https://locus.ufv.br//bitstream/123456789/1417/2/texto%20completo.pdf.txt
https://locus.ufv.br//bitstream/123456789/1417/3/texto%20completo.pdf.jpg
bitstream.checksum.fl_str_mv 1cb09b3dc99fe9425960ca39095e2663
5f0f1eb8aa135858f721c08dd70c4337
ac145e50fb72528f71ea53f6b849c023
bitstream.checksumAlgorithm.fl_str_mv MD5
MD5
MD5
repository.name.fl_str_mv LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)
repository.mail.fl_str_mv fabiojreis@ufv.br
_version_ 1801212854894854144