Submitted by Jaqueline Silva (jtas29@gmail.com) on 2014-09-15T20:06:22Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Karinna Bannach Reis.pdf: 3955087 bytes, checksum: fbfd1023491588a9a5495cd281d72a41 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-09-15T20:06:45Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Karinna Bannach Reis.pdf: 3955087 bytes, checksum: fbfd1023491588a9a5495cd281d72a41 (MD5) / Made available in DSpace on 2014-09-15T20:06:45Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Karinna Bannach Reis.pdf: 3955087 bytes, checksum: fbfd1023491588a9a5495cd281d72a41 (MD5) Previous issue date: 2013-12-18 / Ruta graveolens has been successfully applied for many human diseases treatment and promises a well succeed alternative for plant diseases control because it has also phytoalexins in its composition. The aim of this study was to standardise the R. graveolens liquid extract and evaluate its potential for controlling rice (Oryza sativa) leaf blast (Magnaporthe oryzae). The drug has been characterized, the liquid extract obtained and the methodology for quantifying the standards components, furanocoumarins psoralen and bergapten, validated by high performance liquide chromatography. The components of essential oil obtained from standardized liquid extract were characterized by gas chromatography-mass spectrometry. In a completely randomized design, conducted in artificial hydrophobic surface with three replications and eleven treatments composed of M. oryzae conidial suspension (105 con.ml-1) mixed with R. graveolens standardized extract (0.01 to 0.10 g.mL-1), or furocoumarins psoralen (0.18 to 1.82 μg. mL-1), or bergapten (0.29 to 2.91 μg. mL-1), or water (control). It was evaluated the inhibition of conidial germination and appressorium formation and the median lethal dose (LD50). A second assay, in a completely randomized design in three replications was conducted in greenhouse conditions. It was composed of 21 days old rice plants, which were sprayed with a mixture containing M. oryzae conidial suspension (3x105 con.ml-1) and R. graveolens extract, without dilution, or furocoumarins psoralen (18.26 μg. mL-1), or bergapten (29.14 μg. mL-1), or water (control). Nine days after inoculation, leaf blast severity was scored with a diagrammatic scale, the data were statistically analyzed and means compared. The standardized plant extract inhibited M. oryzae conidial germination (LD50=0.237mg) and appressorium formation (LD50=0.121 mg) up to 100% and reduced 80.84% of leaf blast. By fluorescence microscopy it was possible to observe that standardized plant extract did not damage M. oryzae cell wall and plasma membrane, indicating another type of interaction to inhibit conidia development. Isolated standards furanocoumarins psoralen and bergapten did not inhibit conidial germination and appressorium formation and reduce leaf blast severity proportionally, suggesting that synergistic interactions between extract and essential oil components were responsible for the success of R. graveolens in suppressing rice disease, making it an alternative to compose rice blast management. / Ruta graveolens é utilizada com sucesso no tratamento de diversas patologias humanas e possui potencial para o controle alternativo de fitopatógenos, pois também apresenta fitoalexinas em sua composição. O objetivo deste estudo foi padronizar o extrato vegetal líquido de R. graveolens e avaliar seu potencial para o controle da brusone foliar. O material vegetal foi caracterizado, o extrato líquido obtido e a metodologia para a quantificação dos padrões psoraleno e bergapteno foi validada por cromatografia líquida de alta eficiência. Os componentes do óleo essencial obtidos a partir do extrato líquido padronizado foram caracterizados por cromatografia gasosa acoplada à espectrometria de massas. No ensaio conduzido em superfície hidrofóbica artificial, com três repetições e onze tratamentos, 10 μL da suspensão de conídios de Magnaporthe oryzae (105 con/mL) foram misturadas com o extrato vegetal padronizado (0,01 a 0,10 g/mL), ou as furanocumarinas psoraleno (0,18 a 1,82 μg/mL) e bergapteno (0,29 a 2,91 μg/mL), ou água (controle). Avaliou-se a inibição da germinação conidial e da formação dos apressórios determinando-se a dose letal capaz de inibir 50% dos indivíduos (DL50). No experimento conduzido em casa de vegetação e com três repetições, plantas de arroz com 21 dias após plantio foram pulverizadas com uma suspensão de conídios de M. oryzae (3x105 com/mL) misturada com extrato vegetal padronizado sem diluição, ou psoraleno (18,26 μg/mL), ou bergapteno (29,14 μg/mL), ou água (controle). Após nove dias da inoculação, avaliou-se a severidade de brusone foliar com uma escala de 10 graus, os dados foram analisados estatisticamente e as médias comparadas. O extrato vegetal padronizado inibiu a formação de tubo germinativo (DL50= 0,237 mg) e a formação de apressório (DL50= 0,121 mg) de M. oryzae em até 100%, e reduziu a severidade de brusone nas folhas em 80,84%. Através de microscopia de fluorescência, não foi observada ação do extrato padronizado em membrana plasmática e parede celular de M. oryzae, o que indica outro tipo de interação para inibir o desenvolvimento do conídio. Os padrões psoraleno e bergapteno não inibiram proporcionalmente a germinação e a formação de apressório, como também não reduziram a severidade de brusone nas folhas em sua forma isolada, o que sugere que interações sinérgicas entre os diversos componentes do extrato e do óleo essencial de R. graveolens foram responsáveis pelo sucesso do extrato padronizado em suprimir a brusone foliar, tornando-o uma alternativa para compor o manejo de brusone em arroz.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-20T13:36:24Z No. of bitstreams: 3 Dissertação - Amanda Karla Campos da Costa - 2014- (1).pdf: 18691385 bytes, checksum: cf28696cd33b2f5d65437a46dbb90526 (MD5) Dissertação - Amanda Karla Campos da Costa - 2014 - (2).pdf: 6206670 bytes, checksum: daad6a28f3ab47f826be772e7279df1f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-20T13:38:02Z (GMT) No. of bitstreams: 3 Dissertação - Amanda Karla Campos da Costa - 2014- (1).pdf: 18691385 bytes, checksum: cf28696cd33b2f5d65437a46dbb90526 (MD5) Dissertação - Amanda Karla Campos da Costa - 2014 - (2).pdf: 6206670 bytes, checksum: daad6a28f3ab47f826be772e7279df1f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-20T13:38:02Z (GMT). No. of bitstreams: 3 Dissertação - Amanda Karla Campos da Costa - 2014- (1).pdf: 18691385 bytes, checksum: cf28696cd33b2f5d65437a46dbb90526 (MD5) Dissertação - Amanda Karla Campos da Costa - 2014 - (2).pdf: 6206670 bytes, checksum: daad6a28f3ab47f826be772e7279df1f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-11-28 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Several biological activites... / Diversas atividade biológicas são ...

La rue officinale (Ruta graveolens L) est une plante connue comme étant particulièrement riche en métabolites secondaires et produisant notamment des molécules d’intérêt pharmaceutique comme les furocoumarines. Nous avons tenté par une approche de génie métabolique d’augmenter la teneur en furocoumarines produites dans les plantes. La mise en place de telles approches nous a également permis de mieux comprendre les mécanismes de régulation de la voie de biosynthèse des phénylpropanoïdes. Pour atteindre ces objectifs nous avons transformé la rue avec différents gènes placés sous le contrôle d’un promoteur constitutif fort, le promoteur 35S du CaMV. Pour chaque série de transformants nous avons étudié la teneur en furocoumarines et analysé les variations de composés phénylpropanoïdes (rutine, umbelliférone, ferulate, scopolétine). Parallèlement à cette analyse métabolique, une corrélation a été réalisée avec le niveau d’expression des transgènes et de certains endogènes par l’utilisation d’approche de PCR quantitative. Les séries de plantes transgéniques surexprimant les gènes codants pour la Coumaroyl ester 3’-Hydroxylase de rue (CYP98A22) et d’A. thaliana (CYP98A3) présentent toutes les deux une augmentation significative d’une facteur 3 de la teneur en furocoumarines. Par contre si les premières sont caractérisées par une diminution de la production en rutine et en umbelliférone, les secondes présentent une augmentation importante de la teneur en Scopolétine et en umbelliférone. Ces résultats suggèrent la coexistence de deux C3’H chez R. graveolens ayant des fonctions différentes, l’une d’entre elles étant impliquée directement ou non dans la synthèse de scopolétine. Si la transformation génétique de rues avec des gènes de la famille CYP98A induit des modifications du métabolisme secondaire, la surexpression d’un gène spécifique à la voie de biosynthèse des furocoumarines (gène cyp71AJ1, codant pour la psoralène synthase d’A. majus) permet d’augmenter uniquement la teneur en furocoumarines (X4). L’ensemble de ces travaux a permis de montrer l’intérêt d’une approche de génie métabolique pour générer des plantes présentant un intérêt potentiel pour la production de molécules d’intérêts pharmaceutiques / Garden Rue (Ruta graveolens L.) is a plant known as being particularly rich in secondary metabolites and in particular producing molecules of pharmaceutical interest like furocoumarines. By the use of a metabolic engineering approach, we tried to increase the content of furocoumarines produced in these plants but also to better understand the regulation mechanisms of the phenylpropanoïd biosynthesis pathway. To achieve these goals we transformed Ruta plants with various genes placed under the control of a strong constitutive promoter, CaMV 35S promoter. The plants we obtained were analyzed for their ability to overproduce furocoumarines but also other phenylpropanoïds like ferulate, umbelliferone, scopoletine or rutin. Using Real Time PCR experiments, a correlation was carried out with the level of expression of each transgene and several endogenous genes. Plants overexpressing either the Ruta or the Arabidopsis Coumaroyl ester 3 '-Hydroxylase (CYP98A22 and CYP98A3 respectively) display both a significant increase (3 time level) of the furocoumarin. However if the S-98A22 plants are characterized by a reduction in the production of rutin and umbelliferone, S-98A3 transgenic plants display a significant increase scopoletine and umbelliferone content. These results suggest the coexistence of two C3'H having different functions in Ruta. One of them might be involved more specifically in the synthesis of scopoletine. If the transformation of Ruta with genes belonging to the CYP98A family generates an enlarged of the secondary metabolism, we also showed that the overexpression of a gene belonging to the furocoumarins biosynthesis pathway (CYP71AJ1, the psoralen synthase) allowed a specific stimulation. Indeed a 4 time increase of the content of furocouramins was noticed in these transgenic plant lines. This work made it possible to make evidence of the interest of a metabolic engineering approach to generate plants of interest for the production of pharmaceutical molecules

Les furocoumarines sont des phytoalexines offrant un potentiel thérapeutique important. Les travaux présentés ici portent sur les cytochromes P450 participant à leur voie de biosynthèse. Une étude «structure-fonction» de la cinnamate-4-hydroxylase (C4H) a été réalisée pour identifier les déterminants de la faible sensibilité de la C4H de Ruta graveolens (CYP73A32) au psoralène. Deux régions protéiques semblent impliquées dans l’inactivation différentielle entre CYP73A32 et CYP73A1. L’une, entre les résidus 31 et 58, est responsable de l’affinité pour le psoralène. L’autre, entre les résidus 229 et 379, contrôle la vitesse d’inactivation. La caractérisation de nouveaux P450 de la biosynthèse des furocoumarines a été entreprise. D’une part, plusieurs ADNc partiels ont été clonés chez Ruta graveolens. D’autre part, CYP71AJ1 isolé chez Ammi majus, a été caractérisé comme étant une psoralène synthase. La spécificité de CYP71AJ1 pour la marmésine a été approchée par l’étude d’un modèle 3D. Mots clés : cytochrome P450, psoralène synthase, cinnamate-4-Hydroxylase, C4H, métabolite secondaire, Ruta graveolens, Ammi majus, inactivation autocatalytique, furocoumarines, psoralène, (+)-marmésine, Modélisation 3D, (+)-columbianetine / Furocoumarins are phytoalexins known as efficient therapeutic agents. The work reported here focuses on cytochromes P450 involved in their biosynthesis pathway. A “structure-function” study was realized to understand how C4H from Ruta graveolens (CYP73A32) can resist to psoralen mechanism-based inactivation. Two parts of the protein seems involved in the differential susceptibility of CYP73A32 and CYP73A1 to psoralen. The first, between amino acids 31 and 58, defines differential affinity to psoralen. The second between residues 229 and 379 controls inactivation kinetic. The second part of this work was devoted to cloning and identification of new P450 involved in furocoumarin biosynthesis. On the one hand, several partial cDNA were cloned from Ruta graveolens. On the other hand, CYP71AJ1, cloned from Ammi majus, was identified as a psoralen synthase. The specificity of CYP71AJ1 for marmesin was approached by the study of a 3D model.