Sugarcane apoplast liquid modulating the expression of genes in endophytic diazotrophic bacteria Herbaspirillum rubrisubalbicans strain HCC103

Autor: Polese, Val?ria
Přispěvatelé: Baldani, Jos? Ivo, Vidal, M?rcia Soares, Goi, Silvia Regina, Medici, Leornado Oliveira, Schwab, Stefan, Olivares, F?bio Lopes
Jazyk: portugalština
Rok vydání: 2017
Předmět:
Zdroj: Biblioteca Digital de Teses e Dissertações da UFRRJ
Universidade Federal Rural do Rio de Janeiro (UFRRJ)
instacron:UFRRJ
Popis: Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2021-03-23T15:27:38Z No. of bitstreams: 1 2017 - Val?ria Polese.pdf: 2532934 bytes, checksum: a32e0c6bc2ff2d41df8eb5e7473ca4fd (MD5) Made available in DSpace on 2021-03-23T15:27:38Z (GMT). No. of bitstreams: 1 2017 - Val?ria Polese.pdf: 2532934 bytes, checksum: a32e0c6bc2ff2d41df8eb5e7473ca4fd (MD5) Previous issue date: 2017-02-20 Coordena??o e Aperfei?oamento de Pessoal de N?vel Superior - CAPES Alternatives 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. Alternativas que diminuam a necessidade de fertilizantes nitrogenados na cultura da cana-de-a??car, os custos e os danos ao ambiente sem reduzir a produ??o s?o fundamentais para a sustentabilidade da cultura. Dentre essas alternativas destaca-se o uso de bact?rias diazotr?ficas, que s?o capazes de realizar o processo da fixa??o biol?gica de nitrog?nio (FBN) quando em associa??o com os tecidos da planta. Diversas esp?cies de bact?rias s?o capazes de realizar esse processo tais como a Herbaspirillum rubrisubalbicans estirpe HCC103. No entanto, os mecanismos desta intera??o bact?ria-planta ainda s?o pouco conhecidos. Na busca pelo conhecimento da intera??o planta-bact?ria, a gen?mica funcional baseada em an?lises transcript?micas 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 l?quido do apoplasto na express?o global de genes na bact?ria Herbaspirillum rubrisubalbicans estirpe HCC103. Para tanto, o estudo foi dividido em dois cap?tulos. O primeiro cap?tulo teve como objetivo avaliar e selecionar genes normalizadores adequados para estudos de express?o g?nica por RT-qPCR da estirpe HCC103. Neste sentido, foram avaliados os n?veis de express?o de 5 genes candidatos a normalizadores (rpoA, gyrA, dnaG, recA e gmK) e 8 genes alvos envolvidos no metabolismo de carbono, ap?s o crescimento da bact?ria no caldo de 4 variedades de cana-de-a??car (RB867515, SP701143, RB92579 e IACSP95-5000) e de 3 fontes de carbono (aconitato, malato e glicose). A an?lise da estabilidade da express?o g?nica realizada com os programas GeNorm e Normfinder indicou os genes dnaG e gyrA como os mais est?veis e portanto adequados para uso como genes normalizadores nas an?lises de RT-qPCR da estirpe HCC103. O cap?tulo 2 teve por objetivo o estudo da express?o g?nica diferencial (m?todo de RNA-Seq) da estirpe HCC103 cultivada na presen?a do l?quido do apoplasto da cana-de-a??car extra?do de colmos da variedade RB867515 com 12 meses de idade. A estirpe HCC103 foi cultivada em meio JNFb l?quido a 30?C e 150 rpm at? a metade da fase de crescimento exponencial, quando o caldo bacteriano foi divida em 3 por??es equivalentes e a estas adicionado os seguintes tratamentos, respectivamente: 50% de ?gua destilada (MA); 50% de l?quido do apoplasto (MLA); 50% de meio JNFb novo (MM). Duas horas ap?s a aplica??o dos tratamentos e incuba??o a 30?C e 150 rpm, as c?lulas bacterianas foram coletadas, o RNA foi extra?do, as bibliotecas de cDNA constru?das, e o sequenciamento foi realizado na plataforma Ion-Proton. Para a identifica??o dos genes diferencialmente expressos (GDE) foi empregado o pacote de bioinform?tica CLC. A anota??o funcional de GDE baseados na ontologia gen?tica foi realizada com os programas Blast2GO, WebMGA (Classifica??o funcional ? COG). Todos os genes diferencialmente expressos foram anotados atrav?s do programa de bioinform?tica Artemis. Os resultados mostraram que as categorias funcionais 7 Tradu??o, estrutura ribossomal e biog?nese; Transcri??o; Mecanismos de tradu??o de sinal e Transporte de amino?cidos se destacaram em n?mero de genes reprimidos. Em contraste, as categorias de Metabolismo e transporte de carboidrato; de amino?cido, Mecanismos de transdu??o de sinal e Transcri??o apresentaram maior n?mero de genes induzidos nas duas compara??es (vs. MA e vs. MM). Na classe produ??o e convers?o de energia destacam-se genes que codificam para enzima nitrato redutase os quais foram altamente induzidos na presen?a do l?quido do apoplasto em ambas as compara??es. Em conclus?o, os resultados mostraram uma variedade de genes envolvidos na intera??o planta bact?ria em resposta ? presen?a do l?quido do apoplasto, como por exemplo, genes que codificam para prote?nas do sistema de secre??o do tipo 6 (SST6), flagelo e mecanismos de transdu??o de sinais. Este trabalho, pioneiro, dever? apoiar pesquisas futuras sobre a intera??o de Herbaspirillum rubrisubalbicans estirpe HCC103 com a planta de cana-de-a??car e possivelmente permitir a identifica??o de biomarcadores que auxiliem na maximiza??o do potencial biotecnol?gico da bact?ria como biofertilizante em diferentes variedades de cana-de-a??car
Databáze: OpenAIRE
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