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INPA Programa De Pós-Graduação Em Genética, Conservação E Biologia Evolutiva/INPA INSTITUTO NACIONAL DE PESQUISAS DA AMAZÔNIA – INPA Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva/INPA Anotação do transcriptoma parcial de Anopheles (Nyssorhynchus) darlingi Root, 1926 GILSON MARTINS DE AZEVEDO JUNIOR Manaus, Amazonas maio, 2011 i GILSON MARTINS DE AZEVEDO JUNIOR Anotação do transcriptoma parcial de Anopheles (Nyssorhynchus) darlingi Root, 1926 ORIENTADORA: Dra. Míriam Silva Rafael (INPA) Co-orientador: Dr. Renato Vicentini (UNICAMP) Dissertação apresentada ao Instituto Nacional de Pesquisas da Amazônia como parte dos requisitos para obtenção do título de Mestre em Genética, Conservação e Biologia Evolutiva. Manaus, Amazonas maio, 2011 ii Ficha catalográfica A994 Azevedo Junior, Gilson Martins de Anotação do transcriptoma parcial de Anopheles (Nyssorhynchus) darlingi Root, 1926 / Gilson Martins de Azevedo Junior.--- Manaus : [s.n.], 2011. xi, 66 f. : il. color. Dissertação (mestrado)-- INPA, Manaus, 2011 Orientador : Míriam Silva Rafael Co-orientador : Renato Vicentini Área de concentração : Genética, Conservação e Biologia Evolutiva 1. cDNA. 2. Anopheles darlingi. 3. Anopheles gambiae. 4. Aedes aegypti. 5. Culex quinquefasciatus. 6. Transcriptoma. I. Título. CDD 19. ed. 595.770415 Sinopse: Genes expressos de Anopheles darlingi, a partir da Bioinformática foram anotados, analisados e comparados entre genomas de mosquitos da família Culicidae, tais como Anopheles gambiae, Aedes aegypti e Culex quinquefasciatus. Palavras chave: cDNA, Evolução, Anopheles darlingi, Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, Transcriptoma. iii Aos meus pais Gilson e Maria Elzir e ao Lucas, meu filho, Dedico. iv Agradecimentos Agradeço a Deus pelas energias que me foram dadas nos momentos de fraqueza, pelas forças que não me deixaram desistir Agradeço aos meus pais, Gilson e Maria Elzir, por serem meu porto seguro e pelos ensinamentos de vida e aos meus irmãos, Juliano e Ana, pelo companheirismo e incentivo. Minha vó Nelzi, tia Naná e tia Lúcia, enfim, a todos os meus familiares, agradeço! Ao Lucas, meu filho, que contribuiu muitíssimo para a conclusão dessa dissertação, lembrando-me que eu não devo desistir. À professora doutora Míriam Rafael, minha orientadora, por acreditar, confiar em mim, pelos ensinamentos, que possibilitaram o desenvolvimento dessa dissertação. Obrigado! Ao professor doutor Renato Vicentini, pelos ensinamentos no aprendizado de Bioinformática e análise de dados, pela amizade e por co-orientar esse trabalho de dissertação. Ao Programa Curso de Pós-Graduação em Genética, Conservação e Biologia Evolutiva (GCBEv/INPA), Dr. Jorge Porto (Coordenador), Dra. Vera Scarpassa (ex- Coordenadora), membros do Conselho que deram as condições necessárias para a realização do meu mestrado; e ao corpo docente, pelo aprendizado recebido nas disciplinas ministradas. Ao Ministério da Ciência e Tecnologia (MCT)/Instituto Nacional de Pesquisas da Amazônia (INPA), pela infra-estrutura e apoio técnico recebidos para o desenvolvimento dessa dissertação À Kelly pela compreensão, cumplicidade e palavras de apoio e incentivo durante a redação dessa dissertação v Ao professor doutor Wanderli Pedro Tadei, pelo apoio e incentivo logístico que foram decisivos no desenvolvimento deste trabalho. À Giselle Guimarães, grande amiga e colega de trabalho que também contribuiu com a sua experiência acerca do sequenciamento de ESts de An. darlingi. Obrigado pela amizade e pelo apoio! À Alessandra e Tamara, muito obrigado pela presteza nos trabalhos na Secretaria do GCBEv. À Kyara Formiga de Aquino e Jacqueline da Silva Batista pelos auxílios no Laboratório Temático de Biologia Molecular-LTMB/INPA Ao professores doutores Míriam Silva Rafael e Wanderli Pedro Tadei, Letícia Bridi (MS.c), mestrandas Ketlen Nunes de Souza e Giselle Guimarães (Laboratório de Malária e Dengue/INPA), Spártaco Astolfi Filho e Carlos Gustavo Nunes da Silva e aos colegas Rogério Meireles, Enedina, Johnson (Laboratório de DNA do Centro de Apoio Multidisciplinar-CAM/UFAM), Dra. Ildinete (UNB), CENARGEN, Drs. Mauro Carneiro e Felipe da Silva, pela construção e sequenciamento das bibliotecas de ESTs de larvas e adultos de A. darlingi. À CAPES, pela disponibilização da bolsa de estudo para a realização desta dissertação. À colega e amiga Letícia pelas palavras amigas e por ter cedido gentilmente dados sobre mapeamento in situ que foram necessários em análise de sintenia. A todos os colegas de Laboratório, Mellina, Letícia, Giselle, Kethlen, Pedro, Sabrina e Lumara pela cooperação e companheirismo. A todos os técnicos do Laboratório de Vetores da Malária e Dengue, pelos auxílios nas coletas e identificação de mosquitos. Ao Gláubio pelas ajudas e ensinamentos sobre cuidados com os mosquitos. vi Adelina, Maria e Zilá, muito obrigado pela amizade e apoio desde minha chegada a Manaus. Aos colegas da turma de Mestrado/2009, pelos momentos de solidariedade e companheirismo durante os momentos de agonia. Obrigado pelo espírito de coletividade de todos! Aos companheiros de república, Chico Mário, Daniel Fagundes e Marcos Bento, obrigado pela amizade. E a todos que de alguma forma contribuíram, seja com palavras de incentivo, palavras amigas e/ou conhecimentos específicos da área! Se eu fosse citar o nome de todas as pessoas, eu precisaria de mais folhas! Obrigado a todos, mesmo! vii RESUMO Insetos transmissores de doenças têm sido estudados para caracterização de diversos aspectos biológicos e evolutivos. Sequenciamentos de genomas inteiros têm permitido comparações entre sequências gênicas de espécies diferentes, fornecendo dados moleculares. Citam-se os mosquitos Anopheles gambiae (subfamília Anophelinae), principal vetor da malária na África, cujo genoma contém 278.253.050 pares de bases (pb), o Aedes aegypti, transmissor do Dengue, com 1.310.090.344 pb e Culex quinquefasciatus, vetor de arboviroses, com 579.042.118 pb, ambos da (subfamília Culicinae). No Brasil, o Anopheles darlingi (subfamília Anophelinae) é o principal vetor da malária, cuja bibliotecas de ESTs (Expressed SequenceTags) parcial de adultos e larvas previamente obtidas foram analisadas no presente trabalho. Sequências gênicas de An. gambiae, Ae. aegypti e Cx. quinquefasciatus foram analisadas quanto a presença de genes em comum e genes ortólogos, nesses mosquitos em comparação com An. darlingi. ESTs (568 Unigenes) de An. darlingi foram mapeadas virtualmente em cromossômos de An. gambiae. Em seguida, foram realizadas análises de expressão diferencial gênica, o que permitiu quantificar estatisticamente os níveis de expressão de cada gene, no transcriptoma de An. darlingi. As sequências de An. darlingi foram mapeadas nos termos ontológicos do Gene Ontology (GO) e as sequências diferencialmente expressas, com bons valores estatísticos que indicam ortologia, foram analisadas manualmente, segundo a literatura. Os resultados mostraram que, 61% das sequências de An. darlingi são ortólogas em An. gambiae e o mapeamento cromossômico in silico mostrou que os 568 Unigenes de An. darlingi estão representados em 793 regiões cromossômicas do An. gambiae, corroborando estudos evolutivos de que as duas espécies são próximas evolutivamente. As sequências de An. darlingi analisadas no programa GO foram associadas a 1.249 níveis e subníveis de ontologias que descrevem um gene de acordo com sua função e localização celular. Análises de expressão diferencial de alguns produtos gênicos mostraram-se mais intensas em larvas do que em adultos de An. darlingi. Realizaram-se, ainda, a análise filogenética do gene da Glutationa-S-Transferase (GST) de An. darlingi, um gene de resistência a inseticidas bem conservado em termos evolutivos em An. gambiae, Ae. aegypti e Cx. quinquefasciatus. A árvore filogenética agrupou o gene da GST de An. darlingi com An. gambiae como espécies irmãs, estando estas proximamente relacionadas evolutivamente do que em relação ao Ae. aegypti e Cx. quinquefasciatus. O Phlebotomus papatasi, constou como grupo externo, para enraizamento dessa árvore. A anotação manual de ESTs de An. darlingi auxiliou na compreensão da expressão gênica e aspectos evolutivos desse mosquito em relação ao An. gambiae, Ae. aegypti e Cx. quinquefasciatus, além de fornecer dados importantes para estudos posteriores sobre funções gênicas individuais de An. darlingi transmissor da malária no Brasil. viii Abstract Disease-transmitting insects have been studied to characterize several biological and evolutionary aspects. Whole Genome sequencing has been allowing comparisons between the gene sequences of different species, providing molecular data. The mosquitoes Anopheles gambiae (subfamily Anophelinae), the most important vector of malaria in Africa, whose genome contains 278.253.050 base pairs (bp), Aedes aegypti, transmitter of Dengue, with 1.310.090.344 bp and Culex quinquefasciatus, vector of arbovirus, with 579.042.118 bp, both of (subfamily Culicinae) are cited. In Brazil, the Anopheles darlingi (subfamily Anophelinae) is the most important malaria vector, whose partial EST (Expressed Sequences Tags) libraries of adults and larvae previously obtained were analyzed in this study. Genic sequences of An. gambiae, Ae. aegypti and Cx. quinquefasciatus were analyzed for the presence of orthologous genes, in these mosquitoes in comparison with An. darling. An. darlingi’s ESTs (568 Unigenes) were virtually mapped in An. gambiae’s chromosomes. After that, differential gene expressions were analyzed, which allowed to statistically quantify the levels of differential expression of each gene, in the transcriptome
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