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dc.contributor.authorFernandes, Erika da Costa
dc.date.accessioned2023-12-21T18:56:08Z-
dc.date.available2023-12-21T18:56:08Z-
dc.date.issued2021-10-29
dc.identifier.citationFERNANDES, Erika da Costa. Caracterização de transportadores envolvidos na remobilização de nitrato armazenado no vacúolo em arroz. 2021. 102 f. Tese (Doutorado em Fitotecnia) - Instituto de Agronomia, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, 2021.por
dc.identifier.urihttps://rima.ufrrj.br/jspui/handle/20.500.14407/10031-
dc.description.abstractO acúmulo de nitrato (NO3-) nos vacúolos celulares é uma estratégia de extrema importância para suprir o nitrogênio (N) necessário ao desenvolvimento da planta devido as variações de disponibilidade no solo. O transporte desse íon pelo tonoplasto é mediado por proteínas transportadoras específicas, ainda pouco conhecidas. A identificação dessas proteínas permite esclarecer o processo de acúmulo e remobilização de NO3- e delinear estratégias visando aumentar a eficiência do uso de N. O objetivo deste trabalho foi a identificação de genes que codificam proteínas transportadoras responsáveis pela remobilização do NO3- acumulado no vacúolo em arroz. Inicialmente sequências de aminoácidos potencialmente codificantes de proteínas de transporte de NO3- expressas no vacúolo foram reunidas e realizada uma análise filogenética. Em seguida avaliou-se a resposta de expressão destas proteínas frente ao flush de NO3- em um experimento com plantas de arroz (var. Manteiga) em solução nutritiva de Hoagland com 5 mM de N-NO3- por 30 dias, sob um período de supressão de N por 72 horas, seguido do ressuprimento do fornecimento de N. Posteriormente, os genes OsNPF7.8 e OsNPF5.13 foram clonados inseridos em vetor de superexpressão e realizou-se a transformação genética de plantas de arroz (var. Nipponbare) via A. tumefaciens. Dois experimentos foram realizados semelhantes ao inicial com modificação no tempo de cultivo e nas coletas: as linhagens OsNPF7.8 foram cultivadas em dose constante por 20 dias e submetidas a 72h de supressão seguido pelo ressuprimento do fornecimento de NO3- realizando coletas após a supressão e 24h após o ressuprimento. As linhagens OsNPF5.13 foram cultivadas em dose constante por 24 dias e submetidas a supressão de N realizando coletas 24 e 96 h após a supressão. Um total de 71 genes foram utilizados na criação da árvore filogenética que evidenciou a diferenciação entre as sequências codificantes de canais de cloreto (CLC), transportadores de alta afinidade (NRT2) e de baixa afinidade (NPF) com a separação de 8 grupos. Dentre os transportadores de baixa afinidade o grupo III reuniu genes da família NPF5 caracterizadas como atuantes no efluxo de NO3- a partir do vacúolo em A. thaliana, e o grupo IV genes NPF7 com genes caracterizados como atuantes no influxo de NO3- para o vacúolo em arroz. A análise de expressão demonstrou que os genes OsNPF5.12, OsNPF5.13, OsNPF5.15, OsNPF5.16sp1 e OsNF5.17 apresentaram indução de transcrição em resposta a supressão do fornecimento de NO3- na solução associado ao aumento dos teores de NO3- na planta e da atividade da enzima nitrato redutase (NR), indicando relação com o efluxo de nitrato a partir do vacúolo. Por outro lado, o gene OsNPF7.8 apresentou maior resposta ao período de ressuprimento relacionando-o ao influxo do íon. A superexpressão do gene OsNPF5.13 resultou em maior atividade da NR, aumento dos teores de amônio, N-amino e açucares solúveis demonstrando um aumento da estabilidade da assimilação de N. Enquanto as linhagens superexpressando o gene OsNPF7.8 apresentaram aumento dos teores de NO3- na raiz e da atividade da NR, além de aumento dos teores de amônio e N-amino nas folhas, demonstrando alterações no influxo de NO3- nas raízes.por
dc.description.sponsorshipCAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorpor
dc.description.sponsorshipCNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológicopor
dc.description.sponsorshipFAPERJ - Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiropor
dc.formatapplication/pdf*
dc.languageporpor
dc.publisherUniversidade Federal Rural do Rio de Janeiropor
dc.rightsAcesso Abertopor
dc.subjectproteínas de transporte de baixa afinidadepor
dc.subjectNPFpor
dc.subjectOryza sativapor
dc.subjecttransportadores tonoplastopor
dc.subjectLow affinity transportereng
dc.subjecttonoplast transporterseng
dc.titleCaracterização de transportadores envolvidos na remobilização de nitrato armazenado no vacúolo em arrozpor
dc.title.alternativeCharacterization of transport proteins involved in the remobilization of nitrate accumulated in vacuole of riceeng
dc.typeTesepor
dc.description.abstractOtherThe uptake and remobilization of nitrate (NO3-) in cells vacuoles is an extremely important strategy for nitrogen (N) supply to plant development due to variations in soil availability. The transport of this ion across the tonoplast is mediated by specific transport proteins still poorly understood. The identification of these proteins allows to elucidate NO3- accumulation process and outline strategies in order to increase the use efficiency of N. The aim of this study was to identify genes that encode transport proteins responsible for remobilization of NO3- accumulated in the vacuole in rice. Amino acid sequences potentially coding for NO3- transport proteins expressed in vacuole were selected from gene bank and a phylogenetic analysis performed. The relative expression response of these proteins against the NO3- flush was evaluated in an experiment with rice plants (var. Manteiga) in Hoagland nutrient solution with 5 mM N-NO3- for 30 days, the plants were subjected to N suppression for 72 hours, followed by resupply of N. The genes OsNPF7.8 and OsNPF5.13 were cloned, inserted into an overexpression vector and the genetic transformation of rice plants (var. Nipponbare) was performed via A. tumefaciens. Two more experiments were carried out similar to the initial with modification in cultivation time and harvest: OsNPF7.8 strains were cultivated in constant dose for 20 days and submitted to 72h of suppression followed by the resupply of the NO3- harvesting after the suppression and 24h after resupply. The strains OsNPF5.13 were cultivated at constant dose for 24 days and submitted to N suppression, performing sample collections 24 and 96h after suppression. A total of 71 genes were used in the phylogenetic tree that showed differentiation between the coding sequences of chloride channels (CLC), high affinity (NRT2) and low affinity (NPF) transporters with separation of 8 groups. Among the low affinity transporters, group III clustered sequences of the NPF5 family characterized as responsible of NO3- efflux from the vacuole in A. thaliana and group IV show NPF7 genes mostly characterized as acting in NO3- influx to vacuole in rice. The expression analysis showed that OsNPF5.12, OsNPF5.13, OsNPF5.15, OsNPF5.16sp1 and OsNF5.17 genes presented transcription induction in response to NO3- suppression associated with the increase of NO3-levels in plant and nitrate reductase (NR) activity, it indicates a relationship with nitrate efflux from the vacuole. On the other hand, the OsNPF7.8 gene showed greater response to resupply period, relating to ion influx. The overexpression of OsNPF5.13 gene had higher NR activity, increased ammonium, N-amino and soluble sugar contents, demonstrating an increase in the stability of N assimilation. While overexpression of OsNPF7.8 gene showed increased NO3- content in the root and NR activity, as well as increased ammonium and N-amino content in leaves, demonstrating changes in influx of NO3- in the roots.eng
dc.contributor.advisor1Fernandes, Manlio Silvestre
dc.contributor.advisor1ID002.180.573-37por
dc.contributor.advisor1Latteshttp://lattes.cnpq.br/6269004387821466por
dc.contributor.advisor-co1Santos, Leandro Azevedo
dc.contributor.advisor-co1ID983.907.835-68por
dc.contributor.referee1Fernandes, Manlio Silvestre
dc.contributor.referee2Baldani, José Ivo
dc.contributor.referee3Rouws, Luc Felicianus Marie
dc.contributor.referee4Santos, Marilene Hilma dos
dc.contributor.referee5Alves, Rafaela Eloi de Almeida
dc.creator.ID123.2925.307-96por
dc.creator.IDhttps://orcid.org/0000-0003-2960-1881por
dc.creator.Latteshttp://lattes.cnpq.br/3351916397052664por
dc.publisher.countryBrasilpor
dc.publisher.departmentInstituto de Agronomiapor
dc.publisher.initialsUFRRJpor
dc.publisher.programPrograma de Pós-Graduação em Fitotecniapor
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