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Avaliação da expressão de genes relacionados com a susceptibilidade a Bursaphelenchus xylophilus, agente causal da doença da murchidão dos pinheiros (pine wilt disease) em Pinus pinaster Ait e Pinus yunannensis Franch
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A floresta constitui um bem precioso à escala mundial, do ponto de vista ecológico, paisagístico e económico. No entanto, nos últimos tempos tem-se registado um grave declínio de muitas espécies florestais, sendo em Portugal, o pinheiro bravo, Pinus pinaster Ait das espécies mais afetadas. Uma das principais causas, identificada nas últimas décadas, para este declínio, é o nemátode da madeira do pinheiro. O nemátode da madeira do pinheiro (NMP), Bursaphelenchus xylophilus, é um organismo microscópico que causa a doença da murchidão dos pinheiros “pine wilt disease”. Numa tentativa de diminuir as perdas resultantes da doença, o estudo da resistência do hospedeiro é uma das abordagens de investigação que se iniciou recentemente em Portugal, com o objetivo de melhorar a sua resistência e selecionar árvores com menor suscetibilidade à infeção. A identificação de genes, promotores e marcadores moleculares ligados à resistência à doença, que possam vir a ser mais tarde utilizadas na seleção assistida de genótipos com menor suscetibilidade ao NMP, poderá contribuir para acelerar os programas de melhoramento para a resistência a implementar para esta espécie. Nesse sentido, este trabalho teve como principal objetivo determinar os tempos mais relevantes, após inoculação com NMP, a utilizar para ulterior sequenciação em massa, de bibliotecas de cDNA de Pinus pinaster a a espécie Asiática Pinus yunnanensis, utilizada como controle de tolerância/ resistência para a comparação ulterior de transcritomas destas duas espécies em resposta à infeção com NMP. Foi também comparada a distribuição e propagação do nemátode no interior das duas espécies, após inoculação com NMP, em plantas de três anos, através de observações histológicas. Para determinação dos TAI (Tempos Após Inoculação) relevantes, em termos de expressão de genes candidatos de resistência a stresses bióticos, estudou-se a expressão diferencial dos seguintes genes: Thaumatin like protein, Endo-quitinase, Pinosylvin synthase, Superoxide dismutase-like protein e Allene Oxyde Cyclase, através da técnica Real-Time Polymerase Chain Reaction. As observações histológicas permitiram observar que os canais de resina têm maior diâmetro em Pinus pinaster do que em Pinus yunnanensis, foi possível também verificar que, após inoculação, há um aumento acentuado de inclusões de amido na espécie Mediterrânica, em comparação com a espécie Asiática, em que as inclusões não sofrem alteração de número depois da inoculação com NMP. Quanto aos TAI, pode-se concluir que os primeiros tempos após a infeção, respetivamente 6h e 24h são os tempos em que há uma maior expressão diferencial dos genes em estudo e, portanto, os que se elegem para ulterior sequenciação de bibliotecas de cDNA.
The forest is a valuable resource on a global scale, for ecological, economical and landscape purposes. However, recently there has been a serious decline of many forest species, being the maritime pine, Pinus pinaster Ait the most affected species in Portugal. A major cause detected in recent decades for this decline is the pine wood nematode. The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a microscopic organism that causes pine wilt disease. In an attempt to reduce losses caused by this disease, the study of host resistance is one of the research approaches that recently started in Portugal, aiming to improve their resistance and select trees with lower susceptibility to infection. The identification of genes, promoters and molecular markers linked to disease resistance, that can be later used in marker assisted selection of genotypes with reduced susceptibility to NMP may help to accelerate the breeding programs for resistance to be implemented for this species. Taking this into account, this study aimed to determine the most relevant times after inoculation with NMP to be used for subsequent sequencing of cDNA libraries of Pinus pinaster and Pinus yunnanensis an Asian species, used as control of tolerance / resistance for further comparison of transcriptoms for these two species in response to the infection with PWD. The distribution and spread of nematodes was also compared within the two species after inoculation of three years’ old plants with NMP, by histological observations. For the determination of relevant TAI (Time After Inoculation) gene expression of the following genes was studied: Thaumatin like protein, endo-chitinase, Pinosylvin synthase, Superoxide dismutase-like protein and Allene Oxyde cyclase using Real-Time Polymerase Chain Reaction.Histological observations allowed the observation that the resin ducts have a larger diameter in Pinus pinaster, than in Pinus yunnanensis. It was also possible to verify that, after inoculation, there is an increase of starch inclusions in the Mediterranean type, as compared with the Asian species, where the inclusions do not change in number after inoculation with NMP. Regarding the TAI, one can conclude that early times after infection, respectively 6h and 24h are the ones where a greater differential expression of the genes under study was observed and therefore they are chosen for subsequent sequencing of cDNA libraries.
The forest is a valuable resource on a global scale, for ecological, economical and landscape purposes. However, recently there has been a serious decline of many forest species, being the maritime pine, Pinus pinaster Ait the most affected species in Portugal. A major cause detected in recent decades for this decline is the pine wood nematode. The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a microscopic organism that causes pine wilt disease. In an attempt to reduce losses caused by this disease, the study of host resistance is one of the research approaches that recently started in Portugal, aiming to improve their resistance and select trees with lower susceptibility to infection. The identification of genes, promoters and molecular markers linked to disease resistance, that can be later used in marker assisted selection of genotypes with reduced susceptibility to NMP may help to accelerate the breeding programs for resistance to be implemented for this species. Taking this into account, this study aimed to determine the most relevant times after inoculation with NMP to be used for subsequent sequencing of cDNA libraries of Pinus pinaster and Pinus yunnanensis an Asian species, used as control of tolerance / resistance for further comparison of transcriptoms for these two species in response to the infection with PWD. The distribution and spread of nematodes was also compared within the two species after inoculation of three years’ old plants with NMP, by histological observations. For the determination of relevant TAI (Time After Inoculation) gene expression of the following genes was studied: Thaumatin like protein, endo-chitinase, Pinosylvin synthase, Superoxide dismutase-like protein and Allene Oxyde cyclase using Real-Time Polymerase Chain Reaction.Histological observations allowed the observation that the resin ducts have a larger diameter in Pinus pinaster, than in Pinus yunnanensis. It was also possible to verify that, after inoculation, there is an increase of starch inclusions in the Mediterranean type, as compared with the Asian species, where the inclusions do not change in number after inoculation with NMP. Regarding the TAI, one can conclude that early times after infection, respectively 6h and 24h are the ones where a greater differential expression of the genes under study was observed and therefore they are chosen for subsequent sequencing of cDNA libraries.
Descrição
Tese de mestrado. Biologia (Biologia Celular e Biotecnologia). Universidade de Lisboa, Faculdade de Ciências, 2012
Palavras-chave
Nemátodos Expressão génica Pinheiros Teses de mestrado - 2012