Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana

Detalhes bibliográficos
Autor(a) principal: Chamba, Juan Sebastian Vera
Data de Publicação: 2018
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Biblioteca Digital de Teses e Dissertações da UFRRJ
Texto Completo: https://tede.ufrrj.br/jspui/handle/jspui/5469
Resumo: Nitrogen (N) uptake by plants is a key step for N use efficiency, affecting fresh mass production and yield of grains. The NRT1.1 transceptor of Arabidopsis thaliana was identified as a molecular signal of nitrate uptake (NO3-). In rice, three orthologs of the NRT1.1 named OsNRT1.1A, OsNRT1.1B and OsNRT1.1C, were identified. The objective of this work was to evaluate the overexpression of the genes OsNRT1.1A, OsNRT1.1B and OsNRT1.1C in Arabidopsis thaliana chl1-5 mutant plants to restore the transport and signaling capacity of the nitrate lost in the knockout mutant. The transformation process of A. thaliana plants was obtained by floral immersion with strains of Agrobacterium tumefaciens of lineage LBA4404 by the following constructs 35S: OsNRT1.1A: HA, 35S: OsNRT1.1B: HA, 35S: OsNRT1. 1C: HA and 35S: OsNRT1.1sa: HA (promoter: gene: HA tag). Subsequently, the antibiotic kanamycin was used to obtain segregant lineages of the transformation product, being only plants with two copies of the gene was selected for testing the differents levels of gene expressing. To verify the resistance of the mutant plants to the chlorate (NaClO3), the experiment was set up with homozygous plants, and their seeds were germinated on commercial substrate and vermiculite. At 23 days after planting, applications with 12 mM NaClO3 were started. To evaluate altered gene expression by introduction of OsNRT1.1A, OsNRT1.1B, OsNRT1.1C, primers were designed for expression analysis of the high and low affinity NO3- transporters. Two lines of each transformation were used in the experiments, including wild type plants (WT) and chl1-5 mutant plants. The chlorate test showed the ability of OsNRT1.1A, OsNRT1.1B or OsNRT1.1C to nitrate uptake, evidenced by the decrease in fresh mass caused by the reduction of chlorate to chlorite by nitrate reductase, the chlorite a toxic product to cells. The insertion of transporter OsNRT1.1B caused the largest growth reduction in the chlorate test compared to OsNRT1.1A and OsNRT1.1C, aproximating to the same levels of wild-type (WT). Expression analyzes showed that the insertion of OsNRT1.1A, OsNRT1.1B and OsNRT1.1C genes into Arabidopsis thaliana chl1-5 was able to induce the expression of OsNRT2.1 and OsNAR2.1 genes, and the alternative splicing form of OsNRT1 .1A (OsNRT1.1Asa) did not significantly affect the expression of OsNRT2.1 and OsNAR2.1. The results show the ability of orthologs of NRT1.1 in rice (OsNRT1.1A, OsNRT1.1B and OsNRT1.1C) to nitrate uptake and signal to the expression of other nitrate transporters (transceptor), which may affect the nitrogen efficiency and uptake.
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spelling Santos, Leandro Azevedo983.907.835-68Sperandio, Marcus Vin?cius Loss922.605.357-04Santos, Leandro AzevedoVidal, Marcia SoaresSouza, Marco Andr? Alves de018.193.906-16http://lattes.cnpq.br/5325492321812485Chamba, Juan Sebastian Vera2022-03-21T19:44:26Z2018-04-04CHAMBA, Juan Sebastian Vera. Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana. 2018. 40 f. (Mestrado em Agronomia, Ci?ncia do Solo) - Instituto de Agronomia, Universidade Federal Rural do Rio de Janeiro, Serop?dica, 2018.https://tede.ufrrj.br/jspui/handle/jspui/5469Nitrogen (N) uptake by plants is a key step for N use efficiency, affecting fresh mass production and yield of grains. The NRT1.1 transceptor of Arabidopsis thaliana was identified as a molecular signal of nitrate uptake (NO3-). In rice, three orthologs of the NRT1.1 named OsNRT1.1A, OsNRT1.1B and OsNRT1.1C, were identified. The objective of this work was to evaluate the overexpression of the genes OsNRT1.1A, OsNRT1.1B and OsNRT1.1C in Arabidopsis thaliana chl1-5 mutant plants to restore the transport and signaling capacity of the nitrate lost in the knockout mutant. The transformation process of A. thaliana plants was obtained by floral immersion with strains of Agrobacterium tumefaciens of lineage LBA4404 by the following constructs 35S: OsNRT1.1A: HA, 35S: OsNRT1.1B: HA, 35S: OsNRT1. 1C: HA and 35S: OsNRT1.1sa: HA (promoter: gene: HA tag). Subsequently, the antibiotic kanamycin was used to obtain segregant lineages of the transformation product, being only plants with two copies of the gene was selected for testing the differents levels of gene expressing. To verify the resistance of the mutant plants to the chlorate (NaClO3), the experiment was set up with homozygous plants, and their seeds were germinated on commercial substrate and vermiculite. At 23 days after planting, applications with 12 mM NaClO3 were started. To evaluate altered gene expression by introduction of OsNRT1.1A, OsNRT1.1B, OsNRT1.1C, primers were designed for expression analysis of the high and low affinity NO3- transporters. Two lines of each transformation were used in the experiments, including wild type plants (WT) and chl1-5 mutant plants. The chlorate test showed the ability of OsNRT1.1A, OsNRT1.1B or OsNRT1.1C to nitrate uptake, evidenced by the decrease in fresh mass caused by the reduction of chlorate to chlorite by nitrate reductase, the chlorite a toxic product to cells. The insertion of transporter OsNRT1.1B caused the largest growth reduction in the chlorate test compared to OsNRT1.1A and OsNRT1.1C, aproximating to the same levels of wild-type (WT). Expression analyzes showed that the insertion of OsNRT1.1A, OsNRT1.1B and OsNRT1.1C genes into Arabidopsis thaliana chl1-5 was able to induce the expression of OsNRT2.1 and OsNAR2.1 genes, and the alternative splicing form of OsNRT1 .1A (OsNRT1.1Asa) did not significantly affect the expression of OsNRT2.1 and OsNAR2.1. The results show the ability of orthologs of NRT1.1 in rice (OsNRT1.1A, OsNRT1.1B and OsNRT1.1C) to nitrate uptake and signal to the expression of other nitrate transporters (transceptor), which may affect the nitrogen efficiency and uptake.A absor??o de Nitrog?nio (N) pelas plantas ? uma etapa chave para a efici?ncia de uso de N, afetando a produ??o de massa fresca e rendimento de gr?os. O transceptor NRT1.1 de Arabidopsis thaliana foi identificado como sinalizador da absor??o de nitrato (NO3-). Em arroz, tr?s prov?veis ort?logos do transceptor NRT1.1 foram identificados, nomeados de OsNRT1.1A, OsNRT1.1B e OsNRT1.1C. O objetivo deste estudo foi avaliar se a superexpress?o dos genes OsNRT1.1A, OsNRT1.1B e OsNRT1.1C em plantas mutantes chl1-5 de Arabidopsis thaliana (sem o gene NRT1.1) restabelecem a capacidade de transporte e sinaliza??o pelo nitrato perdida no mutante nocauteado. O processo de transforma??o de plantas chl1-5 de A. thaliana foi mediante floral dip com as cepas de Agrobacterium tumefaciens da linhagem LBA4404 mediante as constru??es obtidas 35S:OsNRT1.1A:HA, 35S:OsNRT1.1B:HA, 35S:OsNRT1.1C:HA e 35S:OsNRT1.1sa:HA (promotor:gene:tag de HA). Posteriormente foi utilizado o antibi?tico canamicina para obter as linhagens segregantes produto da transforma??o, sendo que apenas as plantas com duas c?pias do gene foram selecionadas para testar os diferentes n?veis de express?o g?nica das plantas obtidas. Para verificar a resist?ncia das plantas mutantes ao clorato (NaClO3), foi montado o experimento com plantas homozigotas, e suas sementes foram germinadas sobre substrato comercial e vermiculita, aos 23 dias ap?s o plantio foram iniciadas aplica??es com 12 mM de NaClO3. Para avaliar a express?o g?nica alterada pela introdu??o de OsNRT1.1A, OsNRT1.1B ou OsNRT1.1C no mutante chl1-5, foram desenhados iniciadores para an?lise de express?o dos transportadores de NO3- de alta afinidade e baixa afinidade. Foram usadas duas linhagens de cada transforma??o nos experimentos, al?m de plantas tipo silvagem (WT) e plantas mutantes chl1-5. O teste com clorato mostrou a capacidade dos transportadores OsNRT1.1A, OsNRT1.1B ou OsNRT1.1C de absorver nitrato, evidenciado pelo decr?scimo da massa fresca provocado pela redu??o do clorato a clorito pela nitrato redutase, produto t?xico para as c?lulas. A inser??o do transportador OsNRT1.1B causou a maior redu??o de crescimento no teste do clorato em compara??o com OsNRT1.1A e OsNRT1.1C, chegando aos mesmos n?veis de redu??o do crescimento da planta tipo silvestre (WT). As an?lises de express?o mostraram que a inser??o dos genes OsNRT1.1A, OsNRT1.1B e OsNRT1.1C em Arabidopsis thaliana chl1-5 foi capaz de induzir a express?o dos genes OsNRT2.1 e OsNAR2.1, sendo que a forma splicing alternativo de OsNRT1.1A (OsNRT1.1Asa) n?o afetou de maneira significativa a express?o de OsNRT2.1 e OsNAR2.1. Os resultados obtidos mostram a capacidade dos ort?logos de NRT1.1 em arroz (OsNRT1.1A, OsNRT1.1B e OsNRT1.1C) em absorver nitrato e sinalizar para a express?o de outros transportadores de nitrato (transceptor), podendo afetar a efici?ncia de absor??o de nitrog?nio.Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2022-03-21T19:44:26Z No. of bitstreams: 1 2018 - Juan Sebastian Vera Chamba.pdf: 1264896 bytes, checksum: 1fc6c49cecaecd26a16223e2cfcb7e20 (MD5)Made available in DSpace on 2022-03-21T19:44:26Z (GMT). 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dc.title.por.fl_str_mv Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana
dc.title.alternative.eng.fl_str_mv Characterization of the isoforms OsNTR1.1A, OsNTR1.1B and OsNTR1.1C transporters by phenotypic reversion of the chl1-5 mutant of Arabidopsis
title Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana
spellingShingle Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana
Chamba, Juan Sebastian Vera
Transceptor
Nitrog?nio
Oryza sativa
Arabidopsis thaliana
Transceptor
Nitrogen
Agronomia
title_short Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana
title_full Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana
title_fullStr Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana
title_full_unstemmed Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana
title_sort Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana
author Chamba, Juan Sebastian Vera
author_facet Chamba, Juan Sebastian Vera
author_role author
dc.contributor.advisor1.fl_str_mv Santos, Leandro Azevedo
dc.contributor.advisor1ID.fl_str_mv 983.907.835-68
dc.contributor.advisor-co1.fl_str_mv Sperandio, Marcus Vin?cius Loss
dc.contributor.advisor-co1ID.fl_str_mv 922.605.357-04
dc.contributor.referee1.fl_str_mv Santos, Leandro Azevedo
dc.contributor.referee2.fl_str_mv Vidal, Marcia Soares
dc.contributor.referee3.fl_str_mv Souza, Marco Andr? Alves de
dc.contributor.authorID.fl_str_mv 018.193.906-16
dc.contributor.authorLattes.fl_str_mv http://lattes.cnpq.br/5325492321812485
dc.contributor.author.fl_str_mv Chamba, Juan Sebastian Vera
contributor_str_mv Santos, Leandro Azevedo
Sperandio, Marcus Vin?cius Loss
Santos, Leandro Azevedo
Vidal, Marcia Soares
Souza, Marco Andr? Alves de
dc.subject.por.fl_str_mv Transceptor
Nitrog?nio
Oryza sativa
Arabidopsis thaliana
topic Transceptor
Nitrog?nio
Oryza sativa
Arabidopsis thaliana
Transceptor
Nitrogen
Agronomia
dc.subject.eng.fl_str_mv Transceptor
Nitrogen
dc.subject.cnpq.fl_str_mv Agronomia
description Nitrogen (N) uptake by plants is a key step for N use efficiency, affecting fresh mass production and yield of grains. The NRT1.1 transceptor of Arabidopsis thaliana was identified as a molecular signal of nitrate uptake (NO3-). In rice, three orthologs of the NRT1.1 named OsNRT1.1A, OsNRT1.1B and OsNRT1.1C, were identified. The objective of this work was to evaluate the overexpression of the genes OsNRT1.1A, OsNRT1.1B and OsNRT1.1C in Arabidopsis thaliana chl1-5 mutant plants to restore the transport and signaling capacity of the nitrate lost in the knockout mutant. The transformation process of A. thaliana plants was obtained by floral immersion with strains of Agrobacterium tumefaciens of lineage LBA4404 by the following constructs 35S: OsNRT1.1A: HA, 35S: OsNRT1.1B: HA, 35S: OsNRT1. 1C: HA and 35S: OsNRT1.1sa: HA (promoter: gene: HA tag). Subsequently, the antibiotic kanamycin was used to obtain segregant lineages of the transformation product, being only plants with two copies of the gene was selected for testing the differents levels of gene expressing. To verify the resistance of the mutant plants to the chlorate (NaClO3), the experiment was set up with homozygous plants, and their seeds were germinated on commercial substrate and vermiculite. At 23 days after planting, applications with 12 mM NaClO3 were started. To evaluate altered gene expression by introduction of OsNRT1.1A, OsNRT1.1B, OsNRT1.1C, primers were designed for expression analysis of the high and low affinity NO3- transporters. Two lines of each transformation were used in the experiments, including wild type plants (WT) and chl1-5 mutant plants. The chlorate test showed the ability of OsNRT1.1A, OsNRT1.1B or OsNRT1.1C to nitrate uptake, evidenced by the decrease in fresh mass caused by the reduction of chlorate to chlorite by nitrate reductase, the chlorite a toxic product to cells. The insertion of transporter OsNRT1.1B caused the largest growth reduction in the chlorate test compared to OsNRT1.1A and OsNRT1.1C, aproximating to the same levels of wild-type (WT). Expression analyzes showed that the insertion of OsNRT1.1A, OsNRT1.1B and OsNRT1.1C genes into Arabidopsis thaliana chl1-5 was able to induce the expression of OsNRT2.1 and OsNAR2.1 genes, and the alternative splicing form of OsNRT1 .1A (OsNRT1.1Asa) did not significantly affect the expression of OsNRT2.1 and OsNAR2.1. The results show the ability of orthologs of NRT1.1 in rice (OsNRT1.1A, OsNRT1.1B and OsNRT1.1C) to nitrate uptake and signal to the expression of other nitrate transporters (transceptor), which may affect the nitrogen efficiency and uptake.
publishDate 2018
dc.date.issued.fl_str_mv 2018-04-04
dc.date.accessioned.fl_str_mv 2022-03-21T19:44:26Z
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 CHAMBA, Juan Sebastian Vera. Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana. 2018. 40 f. (Mestrado em Agronomia, Ci?ncia do Solo) - Instituto de Agronomia, Universidade Federal Rural do Rio de Janeiro, Serop?dica, 2018.
dc.identifier.uri.fl_str_mv https://tede.ufrrj.br/jspui/handle/jspui/5469
identifier_str_mv CHAMBA, Juan Sebastian Vera. Caracteriza??o dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da revers?o fenot?pica do mutante chl1-5 de Arabidopsis thaliana. 2018. 40 f. (Mestrado em Agronomia, Ci?ncia do Solo) - Instituto de Agronomia, Universidade Federal Rural do Rio de Janeiro, Serop?dica, 2018.
url https://tede.ufrrj.br/jspui/handle/jspui/5469
dc.language.iso.fl_str_mv por
language por
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