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Caracterização Do Transcriptoma E Parede Celular De Três Espécies De Eucalyptus Com Importância Industrial.”

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Caracterização Do Transcriptoma E Parede Celular De Três Espécies De Eucalyptus Com Importância Industrial.” MARCELA MENDES SALAZAR “CARACTERIZAÇÃO DO TRANSCRIPTOMA E PAREDE CELULAR DE TRÊS ESPÉCIES DE EUCALYPTUS COM IMPORTÂNCIA INDUSTRIAL.” CAMPINAS 2012 ii iii Aos meus pais, Sidnei Salazar e Regina Salazar, e ao meu noivo Ivan, dedico. À minha avó Olga, ofereço. iv AGRADECIMENTOS Ao professor Dr. Gonçalo A. G. Pereira por me conceder a oportunidade e orientação. À FAPESP pelo apoio financeiro. À International Paper do Brasil pelo apoio financeiro e disponibilização do material de estudo. À UNICAMP e à pós-graduação do Instituto de Biologia por toda estrutura e pelo ótimo nível do curso de doutorado. A todos do Laboratório de Genômica e Expressão (LGE) da UNICAMP, especialmente ao grupo de pesquisa do Eucalipto e aos bioinformatas Marcelo e Leandro pelas constribuições para esse trabalho. E aos meus amigos Alinne, Bruna, Silvia, Vanessa, Desis e Bruno pelo companherismo e por tornar tudo mais fácil. À Eli, Sil, D.a Ernê, Mari, Welbe e Bruno por toda a ajuda e prestatividade. A todos do Complex Carbohydrate Reserch Center (CCRC), especialmente à Sivakumar Pattathil e Dr. Michael G. Hahn e também ao pessoal do LAFIECO (USP), em especial Adriana Grandis e Dr. Marcos Buckeridge pelas preciosas contribuições. A todas as minhas amigas e amigos de Campinas e Goiânia que são tão especiais e fundamentais. Agradeço à minha família, em especial meus pais pelo esforço, por todo carinho e dedicação nesses anos de doutorado e nos que precederam e que me fizeram chegar até aqui. Ao meu noivo Ivan pela amizade, companherismo e amor que tornou essa conquista muito especial. Agradeço, enfim, a Deus por permitir que todos esses “grandes ombros” estivessem sempre por perto para que eu pudesse me apoiar! v Sumário Lista de Tabelas ............................................................................................................................ 3 Lista de Figuras ............................................................................................................................. 4 Figures list ..................................................................................................................................... 5 RESUMO....................................................................................................................................... 6 ABSTRACT…………………………………………………………………………………………………6 1. Introdução geral .................................................................................................................... 8 2. Objetivos ............................................................................................................................... 9 3. Revisão Bibliográfica ........................................................................................................... 10 3.1 O gênero Eucalyptus ..................................................................................................................... 10 3.2 O Setor Florestal ............................................................................................................................ 12 3.3 Genômica do Eucalipto.................................................................................................................. 14 3.4 Xilogênese e parede celular .......................................................................................................... 15 3.5 Biossíntese dos componentes da parede celular .......................................................................... 18 4. Referências ......................................................................................................................... 29 CAPÍTULO 1: Xylem transcription profiles indicate potential metabolic responses for economically relevant characteristics of Eucalyptus species ........................................................................... 35 Abstract: ...................................................................................................................................... 35 Background: ................................................................................................................................ 37 Results and Discussion: .............................................................................................................. 39 Xylem RNA sequencing ....................................................................................................................... 39 Construction of a reference sequence database (EUCANEXT) .......................................................... 40 Expression analysis.............................................................................................................................. 41 Functional analysis ............................................................................................................................... 46 Conclusion:.................................................................................................................................. 54 Methodology: ............................................................................................................................... 55 List of abbreviations .................................................................................................................... 57 Authors' contributions .................................................................................................................. 57 Authors' information .................................................................................................................... 58 Acknowledgements ..................................................................................................................... 58 References .................................................................................................................................. 58 CAPÍTULO 2: Caracterização da parede celular secundária de três espécies de Eucalyptus .. 65 5. Introdução ........................................................................................................................... 65 6. Metodologia: ........................................................................................................................ 67 7. RESULTADOS E DISCUSSÃO .......................................................................................... 74 8. Conclusão ........................................................................................................................... 87 9. Referências ......................................................................................................................... 89 CONCLUSÃO GERAL: ............................................................................................................... 92 ANEXOS ..................................................................................................................................... 94 CAPÍTULO 1.2: Avaliação do efeito da super-expressão de genes chave de Eucalyptus em planta modelo (Arabidopsis thaliana)................................................................................................... 107 10. Introdução ......................................................................................................................... 107 11. Metodologia ....................................................................................................................... 109 12. Resultados Preliminares ................................................................................................... 114 13. Perspectivas ...................................................................................................................... 117 14. Referências bibliográficas ................................................................................................. 117 2 Lista de Tabelas Tabela 1: Rendimento e rotação das florestas plantadas no setor de papel e celulose (adaptado de Bracelpa, 2011) ...............................................................................................................................…..13 Tabela 2: Porcentagem e desvio padrão dos resíduos insolúveis em álcool tratados com DMSO ........... 76 Tabela 3: Primers utilizados para avaliação da expressão por Real-T im e P C R ...................................... 112 Tabela 4: Resumo do andamento dos experimentos para obtenção de transformantes de A. thaliana.. 116 3 Lista de Figuras Figura 1: Histologia para demonstrar as principais estruturas que compõem o córtex vascular ............... 17 Figura 2: Estrutura da parede celular de plantas, evidenciando as diversas camadas que formam a parede celular secundária. .................................................................................................................. 18 Figura 3: Representação esquemática da parede celular secundária lignificada. ..................................... 18 Figura 4: Diferentes níveis de organização das cadeias de de celulose. ................................................... 19 Figura 5: Síntese de microfibras de celulose pelos complexos de celulose sintase presentes na membrana plasmática da célula. ......................................................................................................... 20 Figura 6: Modelo hipotético da sacarose sintase associada ao complexo celulose sintase. ..................... 21 Figura 7: Monossacarídeos constituintes de hemiceluloses e pectinas. .................................................... 22 Figura 8: A. Estrutura dos principais tipos de hemicelulose.. ..................................................................... 22
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