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dc.contributor.authorPolese, Valéria
dc.date.accessioned2023-12-21T18:55:49Z-
dc.date.available2023-12-21T18:55:49Z-
dc.date.issued2017-02-20
dc.identifier.citationPolese, Valéria. O líquido do apoplasto de cana-de-açúcar modulando a expressão de genes na bactéria diazotrófica endofítica Herbaspirillum rubrisubalbicans estirpe HCC103. 2017. 148 f. Tese (Programa de Pós-Graduação em Fitotecnia) - Universidade Federal Rural do Rio de Janeiro, Seropédica-RJ.por
dc.identifier.urihttps://rima.ufrrj.br/jspui/handle/20.500.14407/9986-
dc.description.abstractAlternativas que diminuam a necessidade de fertilizantes nitrogenados na cultura da cana-de-açúcar, os custos e os danos ao ambiente sem reduzir a produção são fundamentais para a sustentabilidade da cultura. Dentre essas alternativas destaca-se o uso de bactérias diazotróficas, que são capazes de realizar o processo da fixação biológica de nitrogênio (FBN) quando em associação com os tecidos da planta. Diversas espécies de bactérias são capazes de realizar esse processo tais como a Herbaspirillum rubrisubalbicans estirpe HCC103. No entanto, os mecanismos desta interação bactéria-planta ainda são pouco conhecidos. Na busca pelo conhecimento da interação planta-bactéria, a genômica funcional baseada em análises transcriptômicas tem sido utilizada explorando o papel dos exsudatos radiculares, da seiva vegetal e do fluido do apoplasto. O presente trabalho teve por objetivo avaliar o papel do líquido do apoplasto na expressão global de genes na bactéria Herbaspirillum rubrisubalbicans estirpe HCC103. Para tanto, o estudo foi dividido em dois capítulos. O primeiro capítulo teve como objetivo avaliar e selecionar genes normalizadores adequados para estudos de expressão gênica por RT-qPCR da estirpe HCC103. Neste sentido, foram avaliados os níveis de expressão de 5 genes candidatos a normalizadores (rpoA, gyrA, dnaG, recA e gmK) e 8 genes alvos envolvidos no metabolismo de carbono, após o crescimento da bactéria no caldo de 4 variedades de cana-de-açúcar (RB867515, SP701143, RB92579 e IACSP95-5000) e de 3 fontes de carbono (aconitato, malato e glicose). A análise da estabilidade da expressão gênica realizada com os programas GeNorm e Normfinder indicou os genes dnaG e gyrA como os mais estáveis e portanto adequados para uso como genes normalizadores nas análises de RT-qPCR da estirpe HCC103. O capítulo 2 teve por objetivo o estudo da expressão gênica diferencial (método de RNA-Seq) da estirpe HCC103 cultivada na presença do líquido do apoplasto da cana-de-açúcar extraído de colmos da variedade RB867515 com 12 meses de idade. A estirpe HCC103 foi cultivada em meio JNFb líquido a 30°C e 150 rpm até a metade da fase de crescimento exponencial, quando o caldo bacteriano foi divida em 3 porções equivalentes e a estas adicionado os seguintes tratamentos, respectivamente: 50% de água destilada (MA); 50% de líquido do apoplasto (MLA); 50% de meio JNFb novo (MM). Duas horas após a aplicação dos tratamentos e incubação a 30°C e 150 rpm, as células bacterianas foram coletadas, o RNA foi extraído, as bibliotecas de cDNA construídas, e o sequenciamento foi realizado na plataforma Ion-Proton. Para a identificação dos genes diferencialmente expressos (GDE) foi empregado o pacote de bioinformática CLC. A anotação funcional de GDE baseados na ontologia genética foi realizada com os programas Blast2GO, WebMGA (Classificação funcional – COG). Todos os genes diferencialmente expressos foram anotados através do programa de bioinformática Artemis. Os resultados mostraram que as categorias funcionais 7 Tradução, estrutura ribossomal e biogênese; Transcrição; Mecanismos de tradução de sinal e Transporte de aminoácidos se destacaram em número de genes reprimidos. Em contraste, as categorias de Metabolismo e transporte de carboidrato; de aminoácido, Mecanismos de transdução de sinal e Transcrição apresentaram maior número de genes induzidos nas duas comparações (vs. MA e vs. MM). Na classe produção e conversão de energia destacam-se genes que codificam para enzima nitrato redutase os quais foram altamente induzidos na presença do líquido do apoplasto em ambas as comparações. Em conclusão, os resultados mostraram uma variedade de genes envolvidos na interação planta bactéria em resposta à presença do líquido do apoplasto, como por exemplo, genes que codificam para proteínas do sistema de secreção do tipo 6 (SST6), flagelo e mecanismos de transdução de sinais. Este trabalho, pioneiro, deverá apoiar pesquisas futuras sobre a interação de Herbaspirillum rubrisubalbicans estirpe HCC103 com a planta de cana-de-açúcar e possivelmente permitir a identificação de biomarcadores que auxiliem na maximização do potencial biotecnológico da bactéria como biofertilizante em diferentes variedades de cana-de-açúcarpor
dc.description.sponsorshipCoordenação e Aperfeiçoamento de Pessoal de Nível Superior - CAPESpor
dc.formatapplication/pdf*
dc.languageporpor
dc.publisherUniversidade Federal Rural do Rio de Janeiropor
dc.rightsAcesso Abertopor
dc.subjectFixação biológica de nitrogêniopor
dc.subjectHerbaspirillumpor
dc.subjecttranscriptomapor
dc.subjectRNA-Seq.por
dc.subjectBiological fixation of nitrogeneng
dc.subjectHerbaspirillumeng
dc.subjecttranscriptomeeng
dc.subjectRNA-Seqeng
dc.titleO líquido do apoplasto de cana-de-açúcar modulando a expressão de genes na bactéria diazotrófica endofítica Herbaspirillum rubrisubalbicans estirpe HCC103por
dc.title.alternativeSugarcane apoplast liquid modulating the expression of genes in endophytic diazotrophic bacteria Herbaspirillum rubrisubalbicans strain HCC103eng
dc.typeTesepor
dc.description.abstractOtherAlternatives that reduce the need for nitrogen fertilizers in the sugar cane crop, costs and damages to the environment without reducing production are fundamental to the sustainability of the crop. Among these alternatives, we highlight the use of diazotrophic bacteria, which are capable of performing the biological nitrogen fixation (BNF) process when in association with plant tissues. Several species of bacteria are able to perform this process, such as the Herbaspirillum rubrisubalbicans HCC103 strain. However, the mechanisms of this bacterium-plant interaction are still poorly understood. In the search for knowledge of plant-bacterial interaction, functional genomics based on transcriptomic analysis has been used exploring the role of root exudates, vegetable sap and apoplast fluid. The objective of the present work was to evaluate the role of apoplast liquid in the global expression of genes in the bacterium Herbaspirillum rubrisubalbicans strain HCC103. For that, the study was divided into two chapters. The first chapter aimed to evaluate and select suitable normalizing genes for RT-qPCR gene expression studies of strain HCC103. In this sense, the expression levels of 5 normalizing candidate genes (rpoA, gyrA, dnaG, recA and gmK) and 8 target genes involved in carbon metabolism were evaluated after growth of the bacteria in the broth of 4 sugarcane varieties (RB867515, SP701143, RB92579 and IACSP95-5000) and 3 carbon sources (Aconite, Malate and Glucose). Gene expression stability analysis performed with the GeNorm and Normfinder programs indicated the dnaG and gyrA genes as the most stable and therefore suitable for use as normalizing genes in the RT-qPCR analysis of the HCC103 strain. Chapter 2 had the objective of study the differential gene expression (RNA-Seq method) of the strain HCC103 grown in the presence of sugarcane apoplast liquid extracted from stems of the 12-month-old sugarcane variety RB867515. The HCC103 strain was grown in liquid JNFb medium at 30°C and 150 rpm until the middle of the exponential growth phase, when the bacterial broth was divided into 3 equivalent portions and applied the following treatments: 50% of distilled water (MA); 50% of the apoplast liquid (MLA); 50% of fresh JNFb medium (MM). Two hours after application of the treatments, bacterial cells were collected, RNA was extracted, cDNA libraries were constructed, and sequencing was performed on the Ion-Proton platform. For the identification of differentially expressed genes (DGE) the CLC bioinformatics package was used. The functional annotation of GDE based on the genetic ontology was performed with the Blast2GO, WebMGA (Functional Classification - COG) programs. All differentially expressed genes were annotated using the Artemis bioinformatics program. The results showed that the functional categories Translation, ribosomal structure and biogenesis; Transcription; Signal translation mechanisms; Transport of amino acids showed the highest number of repressed genes. In contrast, the categories of metabolism and carbohydrate transport; 9 Signal transduction and Transcription mechanisms showed the highest number of genes induced in both comparisons (vs. MA and vs. MM). In the class of production and energy conversion, the genes codifying for nitrate reductase enzymewere highly expressed in the presence of applopastic fluid in both comaprisions. In conclusion, the results showed a variety of genes involved in the bacterial plant interaction in response to the presence of apoplast fluid, such as genes encoding SST6 proteins, flagellum and signal transduction mechanisms. This pioneering work should support future research on the interaction of Herbaspirillum rubrisubalbicans strain HCC103 with the sugarcane plant and possibly allow to identify biomarkers that help in maximizing the biotechnological potential of the bacterium as a biofertilizer in different sugarcane varieties.eng
dc.contributor.advisor1Baldani, José Ivo
dc.contributor.advisor1ID538.864.458-87por
dc.contributor.advisor1Latteshttp://lattes.cnpq.br/8391182235603982por
dc.contributor.advisor2Vidal, Márcia Soares
dc.contributor.advisor2ID026.210.947-67por
dc.contributor.advisor2Latteshttp://lattes.cnpq.br/3036544314910366por
dc.contributor.referee1Baldani, José Ivo
dc.contributor.referee2Goi, Silvia Regina
dc.contributor.referee3Medici, Leornado Oliveira
dc.contributor.referee4Schwab, Stefan
dc.contributor.referee5Olivares, Fábio Lopes
dc.creator.ID114.500.507-19por
dc.creator.Latteshttp://lattes.cnpq.br/9217359418793419por
dc.publisher.countryBrasilpor
dc.publisher.departmentInstituto de Agronomiapor
dc.publisher.initialsUFRRJpor
dc.publisher.programPrograma de Pós-Graduação em Fitotecniapor
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