Identificação de genes expressos durante a fase pré-simbiótica da associação ectomicorrízica entre Hydnangium sp. e Eucalyptus grandis e transformação de fungos ectomicorrízicos

• Autor(a) principal: Coelho, Irene da Silva
• Data de Publicação: 2008
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1535
• topic: Ectomicorriza; Hydnangium sp.; Laccaria laccata; Fase pré- simbiótica; Expressão gênica; Hibridação subtrativa supressiva; Protoplastos; Agrobacterium tumefaciens; Transformação; Inibição; Ectomycorrhiza; Hydnangium sp.; Laccaria laccata; Pre- symbiotic phase; Gene expression; Suppressive subtractive hybridization; Protoplasts; Agrobacterium tumefaciens; Transformation; Inhibition; CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS

Gene expression in the ectomycorrhizal fungus Hydnangium sp. during the pre-symbiotic interaction was evaluated using an in vitro mycorrhization technique aiming at constructing a suppressive subtractive cDNA library. The fungus was cultivated in the presence of Eucalyptus grandis roots, with no direct physical contact between both organisms. Genes that code for putative proteins involved in carbohydrate, amino acid, and energy metabolism, transcription and protein synthesis, cellular communication, signal transduction, stress defense, transposons, and proteins related to the biogenesis of cell components were identified. The expression of genes that code for pyruvate dehydrogenase, ATP synthase, a voltage-dependent ion-selective channel, acetyl-CoA acetyl transferase, and hydrophobin was evaluated by quantitative RT-PCR. The differential expression of these genes during the pre-symbiotic interaction confirmed the activation of genes related to beta-oxidation and mitochondrial metabolism, besides validating the suppressive subtractive library obtained. The construction of the subtractive library of Hydnangium sp. will make possible the study of different genes related to mycorrhization, facilitating our understanding of the molecular mechanisms involved in the ectomycorrhizal association. For Hydnangium sp., transformation in the presence of PEG/CaCl2 was necessary to optimize the conditions for the formation and regeneration of protoplasts. The highest production of protoplasts of Hydnangium sp., 1.06 x 107 protoplasts/mL, was obtained after two hours in KCl 0.6 M, in the presence of 20 mg/mL of "Lysing Enzymes" and 0.3 g of two- day old mycelium. For protoplast regeneration, the effects of different osmotic stabilizers, agar concentrations in MNM medium and length of incubation in the hydrolytic preparation were tested. So far, none of the conditions tested allowed the regeneration of Hydnangium sp. protoplasts. For the transformation of Hydnangium sp. mediated by Agrobacterium tumefaciens, several parameters were tested. No transformants were obtained and this was partly explained by the inhibitory effect of Hydnangium sp. on the agrobacteria tested. The transformation of the ectomycorrhizal fungus Laccaria laccata mediated by A. tumefaciens was established with an efficiency 32.6%. The integration of the gene hph in the genome of the transformants was confirmed by the detection of a 690-pb DNA fragment with the expected size. For mutant selection, transformants with only one copy of T-DNA inserted in the genome will be tested for the capacity to form ectomycorrhizas.