Made available in DSpace on 2016-04-26T19:39:32Z (GMT). No. of bitstreams: 1 2009 - Leandro Azevedo Santos.pdf: 3069700 bytes, checksum: dbd1726a5b683e7f290e1829143eacac (MD5) Previous issue date: 2009-06-03 / Funda??o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / To improve nitrogen usage efficiency in plants the rice transcriptional factor OsDof25 was identified and cloned, whose probably orthologe is the maize ZmDof1, already identified and partially characterized. The ZmDof1 was also cloned for comparative analysis with OsDof25, in order to confirm this last one as ZmDof1 orthologe in rice. The constructions for Arabidopsis superexpression of these transcriptional factors were made using the cloning system of gateway technology (Invitrogen), to obtain the expression vectors 35S:ZmDof1:HA and 35S:OsDof25:HA. Lineages with different expression levels of these genes were obtained, but with only one inserted copy. These transgenic lineages when grown in a half strength of MS medium (10mM of NH4 + and 20mM of NO3 -) showed phenotypes with chloroses and growth difficulty; although when they were cultured in soil they showed great vegetative development and delay in the inflorescence emission. When analyzed the gene expression changes induced by the superexpression of these transcriptional factors, it was observed that both genes produced an increase in the expression levels of high and low affinity ammonium transporters (AMT1.1 and AMT2.1, respectively), indicating that these phenotypes may be due to the toxic effect of an excess of ammonium uptake. We also verified an increase of expression for pyruvate kinase (PK1 and PK2), and phosphoenolpyruvate carboxylase (PEPC1 and PEPC2). Pyruvate kinase converts phophoenolpyruvate (PEP) to pyruvate, and phosphoenolpyruvate carboxylase converts PEP to oxalacetate, which is substrate for malate dehydrogenase to form malate. Both pyruvate and malate may feed the Krebs cycle. In addition, there was an increase in the expression of isocitrate dehydrogenase, which is present in the citosol and mitochondria, needed for converting isocitrate to 2- oxoglutarate. Thus, it was hypothesized that the increase of expression levels of these carbon metabolism enzymes was necessary to increase the production of 2-oxoglutarate and, consequently, to reduce the toxic effect of ammonium uptaked. Besides, it was observed an increase of expression levels and activity of glutamate dehydrogenase (GDH). This enzime may work as much in the direction of glutamate amination as in deamination, when the plants were submitted to ammonium excess or carbon limitation conditions, respectively. / Com o objetivo de aumentar a efici?ncia de uso de nitrog?nio (EUN) em plantas, foi identificado e clonado o fator de transcri??o OsDof25 de arroz, cujo prov?vel ort?logo ? o ZmDof1 de milho, j? identificado e parcialmente caracterizado. Tamb?m foi clonado o ZmDof1 para an?lises comparativas com o OsDof25, a fim de comprovar que este ?ltimo ? realmente ort?logo do ZmDof1. As constru??es para superexpress?o destes fatores de transcri??o em Arabidopis foram feitas utilizando o sistema gateway de clonagem para obten??o dos vetores de express?o 35S:ZmDof1:HA e 35S:OsDof25:HA. Foram obtidas linhagens com diferentes n?veis de express?o destes genes, mas com apenas uma inser??o. As linhagens transg?nicas obtidas quando crescidas em meio MS ? for?a i?nica (10mM de NH4 + e 20mM de NO3 -) apresentaram fen?tipos como clorose e dificuldade de desenvolvimento, ao passo que quando cultivadas em solo mostraram desenvolvimento vegetativo mais intenso e atraso para emiss?o da infloresc?ncia. Quando analisadas as modifica??es de express?o g?nica causadas pela superexpress?o destes fatores de transcri??o, observou-se que ambos os fatores de transcri??o provocaram aumento de express?o dos transportadores de am?nio de alta e baixa afinidades (AMT1.1 e AMT2.1 respectivamente), indicando que o fen?tipo observado pode ser devido ao efeito t?xico do excesso de am?nio absorvido. Verificou-se tamb?m aumento de express?o das enzimas piruvato quinase (PK1 e PK2) e fosfoenolpiruvato carboxilase (PEPC1 e PEPC2). A piruvato quinase converte o fosfoenolpurato (PEP) a piruvato, enquanto a fosfoenolpiruvato carboxilase converte o PEP a oxalacetato (OAA) que pode sofrer a??o da malato desidrogenase originando o malato. Ambos os metab?litos, piruvato e malato, alimentam o ciclo de Krebs. Houve tamb?m aumento de express?o da isocitrato desidrogenase, enzima presente na mitoc?ndria (ciclo de Krebs) e no citosol que converte isocitrato a 2-oxoglutarato (2-OG). Assim, ? prov?vel que o aumento da express?o destas enzimas do metabolismo de carbono foi necess?rio para aumentar a produ??o de 2-OG e, por conseguinte, diminuir o efeito t?xico do excesso de am?nio absorvido. Al?m disso, observou-se aumento de express?o e atividade da glutamato desidrogenase (GDH). Essa enzima pode atuar tanto na dire??o da amina??o, quanto na dire??o da desamina??o, em condi??es de excesso de am?nio e/ou sob condi??es de limita??o de carbono nas plantas, respectivamente.