Clonagem e expressão heteróloga da proteína promotora de florescimento
Autor(a) principal: | |
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Data de Publicação: | 2023 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/18811 |
Resumo: | Citrus are perennial woody plants and have different stages of development during their life cycle: germination, juvenile stage, growing stage, and reproductive stage. The juvenile stage is characterized by the absence of flowers and fruits that can vary from 6 to 20 years. The flowering process is composed of a complex and synchronized regulatory network that is shared by different species. The citrus CiFT3 gene and tomato SFT gene, homologous to FLOWERING LOCUS T (FT) from Arabidopsis thaliana, encode an universal mobile signal, known as florigen, that is shared among different species. With the recombinant DNA technology, it’s possible to produce proteins through the expression of cloned genes under the control of promoters. These proteins can be purified in order to obtain a pure biologically active molecule. The objective of this project is the expression and purification of the flowering-promoting proteins of citrus and tomato in a heterologous system, in order to enable the application and evaluation of their effects. The CiFT3 and SFT genes were cloned in the vector pET28a, introduced in competent E. coli BL21(DE3) cells and expressed by inducing with different concentrations of IPTG using different times. So far, the recombinant proteins from citrus and tomato have been purified using immobilized metal affinity chromatography. The best condition for the expression was obtained by using 1mM of IPTG at 37ºC under stirring of 200 rpm for 16 hours (Tov). The expression of the recombinant protein was evaluated by SDS-PAGE, which was present in the soluble fraction. The purified CiFT3 protein had yields ranging from 14 to 153 ng/uL, while the purified SFT protein had yields ranging from 18 to 115 ng/uL. Flowering induction tests were performed in vitro, through root absorption with and without vacuum application, and ex vitro, through leaf and apical meristem absorption, in juvenile citrus, tobacco, and tomato plants. The protein concentrations used in the tests varied from 1 ng/μL to 33,600 ng/μL. Induction tests were not successful in promoting early flowering. A possible explanation for this would be the non-effective translocation of the protein from the leaves, where they were injected, to the apical meristem or significant amounts of flowering-promoting proteins may not have been reached to induce flowering in the plants. To better understand the results obtained, complementary analyses are necessary to investigate the possible causes of non-flowering in the evaluated plants. |
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Soares, Natalia CristinaTakita, Marco Auréliohttp://lattes.cnpq.br/0578612884434541Camargo, Raquel Luciana Boscariolhttp://lattes.cnpq.br/9841465074905675http://lattes.cnpq.br/0793765220447303926aed7e-09e5-49ea-ab40-d12af3ef7caa2023-10-24T15:42:36Z2023-10-24T15:42:36Z2023-08-18SOARES, Natalia Cristina. Clonagem e expressão heteróloga da proteína promotora de florescimento. 2023. Dissertação (Mestrado em Produção Vegetal e Bioprocessos Associados) – Universidade Federal de São Carlos, Araras, 2023. Disponível em: https://repositorio.ufscar.br/handle/ufscar/18811.https://repositorio.ufscar.br/handle/ufscar/18811Citrus are perennial woody plants and have different stages of development during their life cycle: germination, juvenile stage, growing stage, and reproductive stage. The juvenile stage is characterized by the absence of flowers and fruits that can vary from 6 to 20 years. The flowering process is composed of a complex and synchronized regulatory network that is shared by different species. The citrus CiFT3 gene and tomato SFT gene, homologous to FLOWERING LOCUS T (FT) from Arabidopsis thaliana, encode an universal mobile signal, known as florigen, that is shared among different species. With the recombinant DNA technology, it’s possible to produce proteins through the expression of cloned genes under the control of promoters. These proteins can be purified in order to obtain a pure biologically active molecule. The objective of this project is the expression and purification of the flowering-promoting proteins of citrus and tomato in a heterologous system, in order to enable the application and evaluation of their effects. The CiFT3 and SFT genes were cloned in the vector pET28a, introduced in competent E. coli BL21(DE3) cells and expressed by inducing with different concentrations of IPTG using different times. So far, the recombinant proteins from citrus and tomato have been purified using immobilized metal affinity chromatography. The best condition for the expression was obtained by using 1mM of IPTG at 37ºC under stirring of 200 rpm for 16 hours (Tov). The expression of the recombinant protein was evaluated by SDS-PAGE, which was present in the soluble fraction. The purified CiFT3 protein had yields ranging from 14 to 153 ng/uL, while the purified SFT protein had yields ranging from 18 to 115 ng/uL. Flowering induction tests were performed in vitro, through root absorption with and without vacuum application, and ex vitro, through leaf and apical meristem absorption, in juvenile citrus, tobacco, and tomato plants. The protein concentrations used in the tests varied from 1 ng/μL to 33,600 ng/μL. Induction tests were not successful in promoting early flowering. A possible explanation for this would be the non-effective translocation of the protein from the leaves, where they were injected, to the apical meristem or significant amounts of flowering-promoting proteins may not have been reached to induce flowering in the plants. To better understand the results obtained, complementary analyses are necessary to investigate the possible causes of non-flowering in the evaluated plants.Citros são plantas lenhosas perenes e passam por diferentes fases de desenvolvimento durante seu ciclo de vida: germinação, fase juvenil, fase adulta vegetativa e fase reprodutiva. A fase juvenil é caracterizada pela ausência de flores e frutos, a qual pode variar de 6 a 20 anos, dependendo da variedade. O processo de florescimento é regulado por uma complexa e sincronizada rede regulatória que é compartilhada por diferentes espécies. O gene CiFT3 de citros e o gene SFT de tomate são homólogos ao FLOWERING LOCUS T (FT) de Arabidopsis thaliana, o qual codifica um sinal móvel universal, conhecido como florígeno, que é conservado entre diferentes espécies e gêneros. Com a tecnologia do DNA recombinante, é possível a produção de proteínas recombinantes por meio da clonagem do gene de interesse em um vetor de expressão sob o controle de um promotor. Após a expressão, o próximo processo é a purificação para obtenção de uma molécula biologicamente ativa. O objetivo deste projeto foi a expressão e purificação das proteínas promotoras de florescimento de citros e tomate em um sistema heterólogo, de modo a possibilitar a aplicação e avaliação dos seus potenciais de indução de florescimento. Para isso, células competentes de E. coli BL21(DE3) foram transformadas utilizando o vetor pET28a contendo o gene CiFT3 e SFT. Para a indução da expressão, foram utilizadas diferentes concentrações do indutor IPTG em diferentes tempos. A purificação das proteínas foi realizada pelo método de cromatografia de afinidade ao níquel. A melhor condição para a indução de expressão das proteínas foi 1 mM de IPTG a 37ºC, com agitação de 200 rpm, durante 16 horas (Tov). A expressão das proteínas recombinantes foi avaliada por SDS-PAGE, as quais apresentaram-se na fração solúvel. A proteína recombinante CiFT3 apresentou rendimento variando de 14 a 153 ng/uL, enquanto a proteína SFT teve rendimento variando de 18 a 115 ng/uL. Testes de indução do florescimento foram realizados in vitro, através da absorção pelas raízes com e sem aplicação de vácuo, e ex vitro, através da absorção pelas folhas e meristema apical, em plantas juvenis de citros, tabaco e tomate, com concentrações de aplicações variando de 1 ng/μL até 33.600 ng/μL da proteína. Os testes de indução não obtiveram sucesso em promover o florescimento precoce. Uma possível explicação para isso, seria a não translocação efetiva da proteína das folhas, onde foram injetadas, para o meristema apical, ou ainda, quantidades significativas das proteínas promotoras do florescimento nas plantas para induzir o florescimento não foram atingidas. Para compreender melhor os resultados obtidos, análises complementares são necessárias a fim de investigar as possíveis causas do não florescimento nas plantas avaliadas.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)88887.602298/2021-00porUniversidade Federal de São CarlosCâmpus ArarasPrograma de Pós-Graduação em Produção Vegetal e Bioprocessos Associados - PPGPVBA-ArUFSCarAttribution-NonCommercial-NoDerivs 3.0 Brazilhttp://creativecommons.org/licenses/by-nc-nd/3.0/br/info:eu-repo/semantics/openAccessJuvenilidadeFlorígenoExpressão de proteínasCitrosSFTCiFT3Proteína recombinanteJuvenilityFlorigenProtein expressionCitrusRecombinant proteinCIENCIAS AGRARIAS::AGRONOMIAClonagem e expressão heteróloga da proteína promotora de florescimentoCloning and heterologous expression of the flowering promoter proteininfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis60060006e29331-03d2-4150-b6de-99599961ecd3reponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINALDISSERTACAO__Natalia C Soares_PPGPVBA_2023.pdfDISSERTACAO__Natalia C Soares_PPGPVBA_2023.pdfapplication/pdf1764078https://repositorio.ufscar.br/bitstream/ufscar/18811/1/DISSERTACAO__Natalia%20C%20Soares_PPGPVBA_2023.pdf2f476484c170ef1be2de332ec6b6389cMD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8810https://repositorio.ufscar.br/bitstream/ufscar/18811/2/license_rdff337d95da1fce0a22c77480e5e9a7aecMD52TEXTDISSERTACAO__Natalia C Soares_PPGPVBA_2023.pdf.txtDISSERTACAO__Natalia C Soares_PPGPVBA_2023.pdf.txtExtracted texttext/plain133204https://repositorio.ufscar.br/bitstream/ufscar/18811/3/DISSERTACAO__Natalia%20C%20Soares_PPGPVBA_2023.pdf.txt1900334b6d8d9adbaedf56b13b800043MD53ufscar/188112024-05-14 17:17:40.85oai:repositorio.ufscar.br:ufscar/18811Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222024-05-14T17:17:40Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Clonagem e expressão heteróloga da proteína promotora de florescimento |
dc.title.alternative.eng.fl_str_mv |
Cloning and heterologous expression of the flowering promoter protein |
title |
Clonagem e expressão heteróloga da proteína promotora de florescimento |
spellingShingle |
Clonagem e expressão heteróloga da proteína promotora de florescimento Soares, Natalia Cristina Juvenilidade Florígeno Expressão de proteínas Citros SFT CiFT3 Proteína recombinante Juvenility Florigen Protein expression Citrus Recombinant protein CIENCIAS AGRARIAS::AGRONOMIA |
title_short |
Clonagem e expressão heteróloga da proteína promotora de florescimento |
title_full |
Clonagem e expressão heteróloga da proteína promotora de florescimento |
title_fullStr |
Clonagem e expressão heteróloga da proteína promotora de florescimento |
title_full_unstemmed |
Clonagem e expressão heteróloga da proteína promotora de florescimento |
title_sort |
Clonagem e expressão heteróloga da proteína promotora de florescimento |
author |
Soares, Natalia Cristina |
author_facet |
Soares, Natalia Cristina |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/0793765220447303 |
dc.contributor.author.fl_str_mv |
Soares, Natalia Cristina |
dc.contributor.advisor1.fl_str_mv |
Takita, Marco Aurélio |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/0578612884434541 |
dc.contributor.advisor-co1.fl_str_mv |
Camargo, Raquel Luciana Boscariol |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/9841465074905675 |
dc.contributor.authorID.fl_str_mv |
926aed7e-09e5-49ea-ab40-d12af3ef7caa |
contributor_str_mv |
Takita, Marco Aurélio Camargo, Raquel Luciana Boscariol |
dc.subject.por.fl_str_mv |
Juvenilidade Florígeno Expressão de proteínas Citros SFT CiFT3 Proteína recombinante |
topic |
Juvenilidade Florígeno Expressão de proteínas Citros SFT CiFT3 Proteína recombinante Juvenility Florigen Protein expression Citrus Recombinant protein CIENCIAS AGRARIAS::AGRONOMIA |
dc.subject.eng.fl_str_mv |
Juvenility Florigen Protein expression Citrus Recombinant protein |
dc.subject.cnpq.fl_str_mv |
CIENCIAS AGRARIAS::AGRONOMIA |
description |
Citrus are perennial woody plants and have different stages of development during their life cycle: germination, juvenile stage, growing stage, and reproductive stage. The juvenile stage is characterized by the absence of flowers and fruits that can vary from 6 to 20 years. The flowering process is composed of a complex and synchronized regulatory network that is shared by different species. The citrus CiFT3 gene and tomato SFT gene, homologous to FLOWERING LOCUS T (FT) from Arabidopsis thaliana, encode an universal mobile signal, known as florigen, that is shared among different species. With the recombinant DNA technology, it’s possible to produce proteins through the expression of cloned genes under the control of promoters. These proteins can be purified in order to obtain a pure biologically active molecule. The objective of this project is the expression and purification of the flowering-promoting proteins of citrus and tomato in a heterologous system, in order to enable the application and evaluation of their effects. The CiFT3 and SFT genes were cloned in the vector pET28a, introduced in competent E. coli BL21(DE3) cells and expressed by inducing with different concentrations of IPTG using different times. So far, the recombinant proteins from citrus and tomato have been purified using immobilized metal affinity chromatography. The best condition for the expression was obtained by using 1mM of IPTG at 37ºC under stirring of 200 rpm for 16 hours (Tov). The expression of the recombinant protein was evaluated by SDS-PAGE, which was present in the soluble fraction. The purified CiFT3 protein had yields ranging from 14 to 153 ng/uL, while the purified SFT protein had yields ranging from 18 to 115 ng/uL. Flowering induction tests were performed in vitro, through root absorption with and without vacuum application, and ex vitro, through leaf and apical meristem absorption, in juvenile citrus, tobacco, and tomato plants. The protein concentrations used in the tests varied from 1 ng/μL to 33,600 ng/μL. Induction tests were not successful in promoting early flowering. A possible explanation for this would be the non-effective translocation of the protein from the leaves, where they were injected, to the apical meristem or significant amounts of flowering-promoting proteins may not have been reached to induce flowering in the plants. To better understand the results obtained, complementary analyses are necessary to investigate the possible causes of non-flowering in the evaluated plants. |
publishDate |
2023 |
dc.date.accessioned.fl_str_mv |
2023-10-24T15:42:36Z |
dc.date.available.fl_str_mv |
2023-10-24T15:42:36Z |
dc.date.issued.fl_str_mv |
2023-08-18 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
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masterThesis |
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publishedVersion |
dc.identifier.citation.fl_str_mv |
SOARES, Natalia Cristina. Clonagem e expressão heteróloga da proteína promotora de florescimento. 2023. Dissertação (Mestrado em Produção Vegetal e Bioprocessos Associados) – Universidade Federal de São Carlos, Araras, 2023. Disponível em: https://repositorio.ufscar.br/handle/ufscar/18811. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/18811 |
identifier_str_mv |
SOARES, Natalia Cristina. Clonagem e expressão heteróloga da proteína promotora de florescimento. 2023. Dissertação (Mestrado em Produção Vegetal e Bioprocessos Associados) – Universidade Federal de São Carlos, Araras, 2023. Disponível em: https://repositorio.ufscar.br/handle/ufscar/18811. |
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https://repositorio.ufscar.br/handle/ufscar/18811 |
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Universidade Federal de São Carlos Câmpus Araras |
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UFSCar |
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Universidade Federal de São Carlos Câmpus Araras |
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