| Summary: | 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.
|
|---|
