Telchin Licus Licus, Drury 1770) (Lepidoptera: Castiniidae)

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Telchin Licus Licus, Drury 1770) (Lepidoptera: Castiniidae) Universidade de Brasília Instituto de Ciências Biológicas Departamento de Biologia Celular Pós-Graduação em Biologia Molecular Aplicação de estratégias moleculares visando o controle da broca-gigante da cana-de-açúcar (Telchin licus licus, Drury 1770) (Lepidoptera: Castiniidae) Fernando Campos de Assis Fonseca Brasília 2013 Universidade de Brasília Instituto de Ciências Biológicas Departamento de Biologia Celular Pós-Graduação em Biologia Molecular Aplicação de estratégias moleculares visando o controle da broca-gigante da cana-de-açúcar (Telchin licus licus, Drury 1770) (Lepidoptera: Castiniidae) Fernando Campos de Assis Fonseca Orientadora: Profa. Dra. Maria Fátima Grossi de Sá Trabalho apresentado ao Programa de Pós-Graduação em Biologia Molecular do Instituto de Ciências Biológicas da Universidade de Brasília como requisito para obtenção do grau de doutor em Biologia Molecular. Brasília 2013 ii Banca Examinadora Drª. Helaine Carrer Departamento de Ciências Biológicas Escola Superior de agricultura Luiz de Queiroz – Esalq Drª. Maria Cristina Mattar da Silva Embrapa Recursos Genéticos e Biotecnologia Drª. Marlene Teixeira De-Souza Departamento de Biologia Celular Universidade de Brasília Drª. Vera Tavares de Campos Carneiro (membro interno) Embrapa Recursos Genéticos e Biotecnologia Drª. Maria Fátima Grossi de Sá (Orientadora) Embrapa Recursos Genéticos e Biotecnologia Dr. Alexandre Augusto Pereira Firmino (suplente) Embrapa Recursos Genéticos e Biotecnologia Brasília 2013 iii Dedico este trabalho a minha família, em especial, minha mãe Vanessa M. Brasil e meu pai Sérgio Barroso de Assis Fonseca (in memoriam) a quem devo toda a gratidão pela minha formação pessoal e profissional. iv Agradecimentos Agradeço a minha mãe Vanessa por todo o amor, confiança e incentivos para vencer mais esta etapa. Agradeço a meu pai Sérgio por acreditar na minha capacidade e pelas palavras de apoio. Apesar da saudade tenho certeza que acompanha de perto todas as minhas conquistas. Aos meus irmãos Isabel e Leonardo por tudo o que passamos juntos. À minha namorada Mariana por todo o carinho e suporte nos momentos mais doces e também nos mais difíceis. Um grande agradecimento ao Leonardo Macedo, pela amizade e por toda a ajuda prestada com os trabalhos com os insetos, sem a qual este estudo não seria alcançado. Ao Alexandre Firmino, Roberta Coelho e Dijair Jr., por todas as trocas de idéias e experiências que nos permitiram viver um momento único de aprendizado. Ao Felipe Redorat, uma pessoa que me acompanhou em praticamente todos os trabalhos e a quem devo toda a gratidão pela ajuda, companheirismo e amizade. Ao Dr. Wagner Lucena, por me apresentar o mundo da dinâmica molecular e pelas discussões sobre as análises dos dados. Ao Dr. Hugo Molinari por se disponibilizar gentilmente para me ensinar as técnicas de transformação de cana-de-açúcar. À Bárbara Dias, pela amizade e ajuda com a cultura de tecidos. Ao Sr. Luiz Avelar Brandão de Góis, responsável pela captura dos insetos no campo, coleta dos ovos e envio para a Embrapa recursos Genéticos e Biotecnologia. À Lecir Nascimento, Angélica Bússolo, Sineide Rangel e Sheiska Silva, pessoas que realizaram um trabalho especial e que propiciaram a todos o melhor ambiente de trabalho. A todos do laboratório de interação molecular planta praga I: Isabela Lourenço, Janaína de Paula, Itamara Mezzalira, André Júlio, Vívian Miranda, Uriele Almeida, Antônio Américo, Rodrigo Fragoso, Aulus Barbosa, Flávia Mulinari, Eduardo Mulinari, Magda Beneventi, Carlos Pessoa (Cadu), Angelina Basso, Tiago Siqueira, Osmundo Neto, Cristina Mattar, Thiago Golçalves, Anne Winnie, Luís Prestes, Bruna Araújo, Rafael Del Sarto, Ariane v Lacerda, Stephan Dohms, Philippe Lins, Naíra Schwarzstein, Diogo Sá, Hudson Ramos, Gustavo Oliveira, Thales Rocha, Érico Vasconcelos, Caroline Bezerra, Vanessa Olinto, Raquel Sampaio e Jorge Arboleda. Ao pessoal da vibe 4, Erick Nakasu, Mariana Lira, Larissa Tancredi e Natália Lamas, pelos momentos divertidos e sempre bem vindos. À Dra. Fátima Grossi, pela oportunidade e orientação. Graças ao período que passei no laboratório me tornei uma pessoa com uma visão melhor sobre a ciência e com isso cresci profissionalmente e pessoalmente. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), pelo apoio financeiro. À Embrapa Recursos Genéticos e Biotecnologia, pelo fornecimento de toda a estrutura e pelos recursos disponibilizados. À Universidade de Brasília, literalmente minha casa, onde vivi toda a minha vida e pude conhecer o mundo acadêmico. Nunca esquecerei toda a história compartilhada e terei sempre um carinho especial. Ao Programa de Pós-Graduação em Biologia Molecular por todo o conhecimento transmitido também à Ana Hilda Tibet, por toda a atenção e ajuda. vi Sumário Lista de Figuras..................................................................................................................................... 5 Lista de Tabelas .................................................................................................................................... 8 Abreviaturas .......................................................................................................................................... 9 Resumo ................................................................................................................................................ 11 Abstract ............................................................................................................................................... 12 1. Introdução Geral ............................................................................................................................ 13 1.1 Cana-de-açúcar: Características gerais e agronegócio ................................................................ 13 1.2 Insetos-praga da cultura canavieira e alternativas de controle .................................................... 16 1.3 Broca-gigante da cana-de-açúcar ................................................................................................ 18 2. Justificativa ..................................................................................................................................... 22 3. Hipótese ........................................................................................................................................... 23 4. Objetivos .......................................................................................................................................... 23 4.1 Objetivo geral ............................................................................................................................. 23 4.2 Objetivos específicos .................................................................................................................. 23 Capítulo I – Obtenção e análise do transcritoma da broca-gigante da cana-de-açúcar (Telchin licus licus) ..................................................................................................................................................... 24 1. Introdução ....................................................................................................................................... 25 1.1 Sequenciamento de DNA............................................................................................................ 25 1.2 Sequenciamento de segunda geração (Next Generation Sequencing) ........................................ 27 1.3 Pirosequenciamento .................................................................................................................... 28 1.4 Tratamento dos dados obtidos por pirosequenciamento ............................................................. 31 1.5 Transcritoma de insetos .............................................................................................................. 32 1.6 Potenciais alvos para o controle de insetos-praga ....................................................................... 34 1.6.1 Aminopeptidases N .............................................................................................................. 34 2. Material e Métodos ......................................................................................................................... 36 2.1 Obtenção dos insetos e extração de RNA ................................................................................... 36 2.2 Obtenção da biblioteca de cDNA e pirosequenciamento ........................................................... 37 2.3 Pré-processamento das sequências ............................................................................................. 37 2.4 Montagem, anotação e ontologia gênica (GO) ........................................................................... 38 2.5 Comparação da biblioteca de Telchin licus licus contra sequências de Manduca sexta ............. 38 2.6 Sequenciamento do gene da aminopeptidase N1 ........................................................................ 39 2.7 Análise das sequências de aminopeptidases ............................................................................... 40 3. Resultados e Discussão ................................................................................................................... 41 1 3.1 Extração de RNA total ................................................................................................................ 41 3.2 Pirosequenciamento e pré-processamento .................................................................................
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