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Validação De Moléculas De Dsrna Visando O Controle Da Broca-Gigante Da Cana- De-Açúcar (Telchin Licus Licus, Drury 1770) E

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Validação De Moléculas De Dsrna Visando O Controle Da Broca-Gigante Da Cana- De-Açúcar (Telchin Licus Licus, Drury 1770) E Universidade de Brasília Instituto de Ciências Biológicas Departamento de Biologia Celular Pós-Graduação em Biologia Molecular Validação de moléculas de dsRNA visando o controle da broca-gigante da cana- de-açúcar (Telchin licus licus, Drury 1770) e da broca da cana-de-açúcar (Diatraea saccharalis, Fabricius 1794) Daniel David Noriega Vásquez Brasília 2018 Universidade de Brasília Instituto de Ciências Biológicas Departamento de Biologia Celular Pós-Graduação em Biologia Molecular Validação de moléculas de dsRNA visando o controle da broca-gigante da cana- de-açúcar (Telchin licus licus, Drury 1770) e da broca da cana-de-açúcar (Diatraea saccharalis, Fabricius 1794) Daniel David Noriega Vásquez Orientadora: 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 mestre em Biologia Molecular. Brasília 2018 Banca Examinadora Dr. Bergmann Morais Ribeiro (membro interno) Departamento de Biologia Celular Universidade de Brasilia Dr. Erich Yukio Tempel Nakasu (membro externo) Embrapa Hortaliças Drª. Maria Fátima Grossi de Sá (Orientadora) Embrapa Recursos Genéticos e Biotecnologia Dr. Elibio Leopoldo Rech Filho (suplente) Embrapa Recursos Genéticos e Biotecnologia Brasília 2018 Dedico este trabalho para louvor de Deus, meu criador, protetor e o responsável de guiar meu caminho. Assim, como a minha familia, que tem todo o merito pela minha formação pessoal e profissional. Agradecimentos Agradeço em primeiro lugar a Yahweh, meu Deus, criador, senhor, pai, protetor, mentor, refugio, provedor, libertador, guerreiro e amigo. A ele devo tudo o que sou e o que tenho conseguido, quem me deu todos meus talentos, formou meu caráter e me ajuda a superar minhas fraquezas cada dia. Agradeço a minha familia por ser a maior motivação da minha vida. A minha mãe Floripes por me ensinar os caminhos da cautela, a calma e a sabedoria. A meu pai Yorlli por ter me ensinado a nunca desistir, a enfrentar meus medos e a fazer tudo com paixão. A meu irmãozinho Efraín por acreditar sempre em mim e nos meus sonhos. Um agradecimento especial ao meu melhor amigo David Augusto Orduy, o irmão que escolhi, quem me ajudou a controlar meus demônios nos momentos difíceis deste processo, me ensinando a rir da vida e de mim mesmo. Outro grande agradecimento a um amigo extraordinário que conheci durante este processo, Joaquin Felipe Roca, por ter sido o irmão mais velho que nunca tive, por me ensinar a amadurecer pessoal e profissionalmente e finalmente por me aguentar de forma tão corajosa estes dois anos. Aos integrantes do grupo de bioinformática do laboratório de interação molecular planta praga I: à Dra. Saluana Rocha por ser a luz que iluminava os dias cinzas com sua admirável energia, dedicação e paixão. Ao Dr. Muhammed Faheem por me orientar com a parte bioquímica do meu trabalho e finalmente ao meu colega Fabricio Arraes por ter trabalhado incessantemente comigo em todas as análises bioinformáticas e me orientar sempre com a melhor das atitudes. Aos demais integrantes do laboratório de interação molecular planta praga I, que me ajudaram em diferentes momentos deste processo. Especialmente a Thuanne Ribeiro, Leonardo Pepino, Hudson Moura, Clidia Moreira, Daniela Amaral, Niday Nunes, Naldo Silva, François Xavier, Caroline Bournaud, Reneida Mendes e Dijair Souza. À equipe de bioinformática do Cenargen, o Dr. Roberto Togawa e a Dra. Priscila Grynberg pelas orientações nas análises de processamento de sequencias. Às pessoas que cuidadosamente corrigiram minha dissertação: a Dra. Saluana Rocha, a Dra. Cristina Mattar, o Dr. Alexandre Firmino e o meu colega Fabricio Arraes. À Dra. Fátima Grossi, a incansável líder do laboratório de interação molecular planta praga, pela grande oportunidade e orientação, que me ajudaram a crescer profissionalmente e pessoalmente. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), pelo apoio financeiro. À Embrapa Recursos Genéticos e Biotecnologia, pelo fornecimento de toda a estrutura e pelos recursos disponibilizados. À Universidade de Brasília, pelo enriquecimento acadêmico e cultural que obtive desta maravilhosa instituição. Ao Programa de Pós-Graduação em Biologia Molecular por ter me permitido viver esta aventura de fazer um curso de pos-graduação fora do meu país natal. Sumário Lista de Figuras ........................................................................................................................ 4 Lista de Tabelas ........................................................................................................................ 9 Abreviaturas............................................................................................................................ 10 Resumo .................................................................................................................................... 13 Abstract ................................................................................................................................... 14 1. Introdução Geral ................................................................................................................ 15 1.1 Cultura da cana-de-açúcar ............................................................................................... 15 1.2 Cultura canavieira no Brasil............................................................................................ 16 1.3 Controle de pragas na cultura da cana-de-açúcar ........................................................... 17 1.4 Broca da cana-de-açúcar ................................................................................................. 19 1.5 Broca-gigante da cana-de-açúcar .................................................................................... 22 1.6 RNA de interferência como alternativa de controle ....................................................... 26 2. Justificativa ......................................................................................................................... 27 3. Objetivo Geral..................................................................................................................... 28 3.1 Objetivo capítulo I .......................................................................................................... 28 3.2 Objetivo capítulo II ......................................................................................................... 28 Capítulo I - Sequenciamento dos transcritomas de D. saccharalis e T. l. licus, e análise da expressão gênica em diferentes dietas. ..................................................................................... 29 1. Introdução ........................................................................................................................... 30 1.1 Sequenciamento de ácidos nucleicos .............................................................................. 30 1.2 Sequenciamento de segunda geração .............................................................................. 31 1.3 Sequenciamento de RNA ................................................................................................ 31 1.4 Transcritômica em insetos .............................................................................................. 33 1.5 Análise de expressão diferencial de genes ...................................................................... 34 1.6 Estudo da expressão genica em insetos .......................................................................... 36 2. Objetivo geral ...................................................................................................................... 38 2.1 Objetivos específicos ...................................................................................................... 38 3. Material e métodos ............................................................................................................. 39 3.1 Obtenção dos insetos e extração de RNA ....................................................................... 39 3.2 Analise dos transcritomas ............................................................................................... 40 3.2.1 Pré-processamento das sequências ........................................................................... 40 1 3.2.2 Montagem e anotação ............................................................................................... 40 3.2.3 Análise de expressão diferencial .............................................................................. 41 3.2.4 Enriquecimento de termos GO ................................................................................. 41 4. Resultados e discussão ........................................................................................................ 42 4.1 Extração de RNA total .................................................................................................... 42 4.2 Análise dos transcritomas ............................................................................................... 42 4.2.1 Pré-processamento ................................................................................................... 42 4.2.2 Montagem de novo e anotação funcional ................................................................. 43 4.2.3 Expressão diferencial de genes ...............................................................................
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