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Advisor(s)
Abstract(s)
A doença do declínio do sobreiro é um fenómeno complexo que provoca uma
diminuição visível do vigor vegetativo da árvore. O fungo Phytophthora cirmamomi está
associado a esta doença radicular, provocando a morte maciça das raízes finas, reduzindo
acentuadamente a capacidade das árvores para absorver água e substâncias nutritivas do solo,
conduzindo ao enfraquecimento gradual das extremidades dos ramos e à queda das folhas.
As elieitinas são um grupo de holoproteínas altamente conservadas segregadas por
espécies do género Phytophthora (com a excepcção da Phytophthora parasitica var.
nicotianae) e por Pythium vexans. Estes polipeptídeos de baixa massa molecular ( ~ 10,3 kDa),
constituídas por 98 aminoácidos, induzem uma resposta de hipersensibilidade e resistência
sistémica adquirida em espécies de plantas sensíveis.
A função das elieitinas na biologia das espécies Phytophthora ainda não é clara, não se
sabendo se estas proteínas contribuem directamente para a patogenicidade das espécies
Phytophthora.
O presente trabalho foi dividido em três partes. Na primeira parte, extraiu-se o RNA
total de micélio de P. cirmamomi, a partir do qual se sintetizaram e amplificaram, por PCR, os
ORF°s (“Operi Reading Frame ”) dos quatro genes do cluster recorrendo a primers específicos.
Os produtos da amplificação foram sujeitos a uma análise de restrição enzimática.
Na segimda parte, fez-se um estudo da expressão de elieitinas de diferentes isolamentos
de duas espécies de Phytophthora. A análise deste estudo mostrou que o isolamento Pa20 de
P. cirmamomi expressa uma maior quantidade de elieitinas e num intervalo de tempo menor,
em comparação com os isolamentos estudados de P. cambivora.
Na terceira parte, produziu-se B-cinamomina e ot-cinamomina heterólogas. Com o
recurso a células de Pichia pastoris, previamente transformadas com o ORF do gene da
B-cinamomina induziu-se a expressão da respectiva proteína. Os sobrenadantes contendo a
B-cinamomina passaram por mn processo de purificação que incluiu: concentração, diálise,
cromatografia de troca iónica e Fast Protein Liquid Chromatngmphy (FPLC). A análise da
B-cinamomina foi feita através de SDS-PAGE e immunobloting.
Cork oak decline is a complex phenomenon that causes a visible reduction of the vegetative vigour of the tree. The fimgus Phytophthora cinnamomi is associated with this root disease, causing a massive death of fine roots, reducing highly the capacity of trees to absorb water and nutritional substances from soil, leading to typical dieback symptoms (gradual weakness of branches extremities and to the fall of leaves). Elicitins are a group of highly conserved holoproteins secreted by species of Phytophthora genus (with the excepction of the Phytophthora parasitica var. nicotianae) and by Pythium vexans. These polypeptides of low molecular weight (~ 10,3 kDa), composed by 98 aminoacids, induce a hypersensitive-like reaction (HR) and a systemic acquired resistance (SAR) in sensitive plant species. The fimction of elicitins in the biology of the Phytophthora species is not yet entirely understood. It is not clear whether these proteins contribute directly to the pathogenicity of Phytophthora species. The present work was divided in three parts. In the first one, total RNA from mycelium of P. cinnamomi was extracted and used to synthesize and amplify specifically, by PCR, each of the ORF”s (Open Reading Frame) of the four elicitin genes using specific primers. Amplification products have been submitted to an enzymatic restriction analysis. In the second part, the expression of elicitins by different isolates and two species of Phytophthora has been studied. This study showed that P. cinnamomi Pa20 isolate secretes a larger amount of elicitins quicker than the P. cambivora isolates. In the third part, heterologous B-cinamomin and ot-cinamomin have been produced. Using Pichia pastoris cells, previously transformed with the ORF of the [3-cinamomin gene, the expression of the corresponding protein was induced. B-cinamomin was purified from the supernatant by a process that included: concentration, dialysis, ion exchange chromatography and fast protein liquid chromatography (FPLC). Analysis of [5-cinamomin was perfomed using SDS-PAGE and immunobloting.
Cork oak decline is a complex phenomenon that causes a visible reduction of the vegetative vigour of the tree. The fimgus Phytophthora cinnamomi is associated with this root disease, causing a massive death of fine roots, reducing highly the capacity of trees to absorb water and nutritional substances from soil, leading to typical dieback symptoms (gradual weakness of branches extremities and to the fall of leaves). Elicitins are a group of highly conserved holoproteins secreted by species of Phytophthora genus (with the excepction of the Phytophthora parasitica var. nicotianae) and by Pythium vexans. These polypeptides of low molecular weight (~ 10,3 kDa), composed by 98 aminoacids, induce a hypersensitive-like reaction (HR) and a systemic acquired resistance (SAR) in sensitive plant species. The fimction of elicitins in the biology of the Phytophthora species is not yet entirely understood. It is not clear whether these proteins contribute directly to the pathogenicity of Phytophthora species. The present work was divided in three parts. In the first one, total RNA from mycelium of P. cinnamomi was extracted and used to synthesize and amplify specifically, by PCR, each of the ORF”s (Open Reading Frame) of the four elicitin genes using specific primers. Amplification products have been submitted to an enzymatic restriction analysis. In the second part, the expression of elicitins by different isolates and two species of Phytophthora has been studied. This study showed that P. cinnamomi Pa20 isolate secretes a larger amount of elicitins quicker than the P. cambivora isolates. In the third part, heterologous B-cinamomin and ot-cinamomin have been produced. Using Pichia pastoris cells, previously transformed with the ORF of the [3-cinamomin gene, the expression of the corresponding protein was induced. B-cinamomin was purified from the supernatant by a process that included: concentration, dialysis, ion exchange chromatography and fast protein liquid chromatography (FPLC). Analysis of [5-cinamomin was perfomed using SDS-PAGE and immunobloting.