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Arquivo3643 1.Pdf UNIVERSIDADE FEDERAL DE PERNAMBUCO CENTRO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE GENÉTICA PROGRAMA DE PÓS-GRA DUAÇÃO EM GENÉTICA E BIOLOGIA MOLECULAR DISSERTAÇÃO DE MESTRADO ANÁLISE COMPUTACIONAL DE GENES ASSOCIADOS AO METABOLISMO DE FIXAÇÃO DE NITROGÊNIO NO FEIJÃO-CAUPI (Vigna unguiculata) E CANA-DE-AÇÚCAR (Saccharum spp.) GABRIELA SOUTO VIEIRA DE MELLO RECIFE 2009 1 UNIVERSIDADE FEDERAL DE PERNAMBUCO CENTRO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE GENÉTICA PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA E BIOLOGIA MOLECULAR DISSERTAÇÃO DE MESTRADO ANÁLISE COMPUTACIONAL DE GENES ASSOCIADOS AO METABOLISMO DE FIXAÇÃO DE NITROGÊNIO NO FEIJÃO- CAUPI (Vigna unguiculata) E CANA-DE-AÇÚCAR (Saccharum spp.) GABRIELA SOUTO VIEIRA DE MELLO Dissertação apresentada ao Programa de Pós-graduação em Genética e Biologia Molecular da Universidade Federal de Pernambuco como requisito para obtenção do grau de Mestre em Genética pela UFPE Orientadora: Profª. Drª. Ana Maria Benko-Iseppon Co-orientador: Prof. Dr. Tercílio Calsa Júnior RECIFE 2009 2 Mello, Gabriela Souto Vieira de Análise computacional de genes associados ao metabolismo de fixação de nitrogênio no feijão-caupi (Vigna unguiculata) e cana-de-açúcar (Saccharum spp.) / Gabriela Souto Vieira de Mello. – Recife: O Autor, 2009. 160 folhas : il., fig., tab. Dissertação (mestrado) – Universidade Federal de Pernambuco.CCB. Genética, 2009. Inclui bibliografia e anexo. 1. Genética Molecular 2. Bioinformática 3. Feijão-Caupi 4. Cana-de-açúcar I Título. 577.21 CDU (2.ed.) UFPE 572.8 CDD (22.ed.) CCB – 2009-142 3 4 “Mude suas opiniões, sustente seus princípios; troque suas folhas, mas mantenha intacta suas raízes” Victor Hugo 5 Agradecimentos A Deus por tudo e por ter colocado tantas pessoas maravilhosas na minha vida. À Minha mãe, Uitamira pelo suporte moral e financeiro, por estar presente em todos os momentos da minha vida, pelo eterno incentivo e por manter nossa família unida que, junto com meu irmão João, me deram forças para correr atrás de tudo que eu sempre quis. Ao Meu pai, Ricardo José (in memorian), pelo exemplo de vida e força espiritual. À minha avó Irene pelo maravilhoso acolhimento em sua casa. Aos meus padrinhos Roberto Vieira de Mello e Maristela Ferraz por sempre acreditarem em mim. A todos os meus familiares, todos vocês, cada um de uma forma peculiar, iluminaram meu caminho. À minha irmã de coração Petra, que me ensinou tudo com uma enorme paciência, por sempre estar ao meu lado nas piores horas, pelas risadas, pelos maravilhosos momentos de descontração. Por TUDO, sem ela esse trabalho nunca teria sido feito. Aos meus amigos Marcela Randau, Mirella Soares e Moacyr Barreto por terem enchido minha pós-graduação de momentos felizes e descontraídos, os levarei para sempre na minha memória. Ao Túlio pelo apoio incondicional, por sempre me ajudar com uma palavra de conforto e uma idéia, por tanto me fazer rir e principalmente pela eterna paciência. À minha orientadora Profa. Ana Maria Benko-Iseppon pela oportunidade e por me ensinar a acreditar mais em mim. Ao meu co-orientador Tercílio Calsa Júnior pelos ensinamentos. A todos os meus amigos e membros do Laboratório de Genética e Biotecnologia Vegetal que tornaram o andamento desse mais agradável. A Carol e Luís pelos ensinamentos e principalmente a Nina pelas incontáveis ajudas e por sempre me apoiar. Aos professores do Programa de Pós-Graduação em Genética. À CAPES pela bolsa concedida durante o desenvolvimento do projeto. 6 SUMÁRIO Item Página LISTA DE ABREVIATURAS ......... VIII LISTA DE FIGURAS ......... XI LISTA DE TABELAS ......... XII RESUMO ......... XVI ABSTRACT ......... XIII INTRODUÇÃO ......... 15 CAPÍTULO 1. Revisão da literatura ......... 17 1.1. Interações Planta-microoganismo ......... 18 1.2. Fixação Biológica de Nitrogênio (FBN) ......... 19 1.2.1. Importância Econômica e Ambiental ......... 19 1.2.2. Mecanismos da FBN em Angiospermas ......... 21 1.2.3. FBN em Vigna unguiculata ......... 23 1.2.4. FBN em Saccharum sp. ......... 23 1.2.5. Aspectos Genéticos da FBN ......... 24 1.2.6. Principais Nodulinas Primárias ......... 25 1.2.7. Principais Nodulinas Secundárias ......... 32 1.3. O Feijão-Caupi ......... 38 1.3.1. Importância Econômica ......... 38 1.3.2. Origem e Distribuição Geográfica ......... 39 1.3.3. Melhoramento do Feijão-Caupi ......... 40 1.3.4. Aspectos Botânicos e Genéticos ......... 41 1.3.5. Projetos HarvEST, NordEST e CGKB ......... 42 1.4. A Cana-de-Açúcar ......... 44 1.4.1. Importância Econômica ......... 44 1.4.2. Origem e Distribuição Geográfica ......... 45 1.4.3. Melhoramento da Cana-de-Açúcar ......... 45 1.4.4. Aspectos Botânicos e Genéticos ......... 46 1.4.5. Projeto SUCEST ......... 47 1.5. Análise Bioinformática ......... 49 6 1.5.1. Retrospectiva e Aplicações Atuais ......... 49 1.5.2. Bancos de Dados, Ferramentas e Programas ......... 50 2. Referências Bibliográficas ......... 52 CAPÍTULO 2 - Computational Analysis of Genes Associated with ......... 70 Symbiotic Nitrogen Fixation in the Cowpea (Vigna unguiculata) Transcriptome - Artigo a ser enviado para a revista Genetics and Molecular Research. CAPÍTULO 3 - Expression of Nodulins in Sugarcane Transcriptome ......... 115 Revealed by Computational Analysis - Artigo a ser enviado para a revista Genetics and Molecular Research. CONCLUSÕES GERAIS ......... 156 ANEXO 157 7 LISTA DE ABREVIATURAS APC Anaphase Promoter Complex (Complexo Promotor da Anáfase) ATP Adenosina Trifosfato BLAST Basic Local Alignment Search Tool (Ferramenta Básica de Alinhamento Local de Seqüências) CCaMK Calcium/Calmodulin Dependent Protein Kinase-like (Quinase Cálcio- Calmodulina-Dependente CCS52 Cell Cycle Switch Protein (Proteína Interruptora do Ciclo Celular) CD Conserved Domain (Domínio Conservado) CDK Cyclin-Dependent Kinase (Quinase Ciclina-dependente) cDNA Complementary Desoxyribonucleic Acid (Ácido Desoxirribonucléico Complementar) DDBJ DNA Database of Japan (Banco de dados de DNA do Japão) DEFH125 Deficient Homolog 125 (Homólogo Deficiente 125) DMI Does Not Make Infection (Não Realiza a Infecção) DMT Divalent Metal Transporter (Transportador de Metais Divalentes) EMBL European Molecular Biology Laboratory (Laboratório Europeu de Biologiao Molecular) EMBRAPA Empresa Brasileira de Pesquisa Agropecuária ENOD Early Nodulin (Nodulina Primária) EST Expressed Sequence Tag (Etiqueta de Seqüência Expressa) FAPESP Fundação de Amparo à Pesquisa do Estado de São Paulo FBN Fixação Biológica do Nitrogênio GenBank Banco de Genes do NCBI GOGAT Glutamato Sintase VIII 8 GS Glutamina Sintase IITA International Institute of Tropical Agriculture (Instituto Internacional de Agricultura Tropical) KEGG Kyoto Encyclopedia of Genes and Genomes (Enciclopédia de Genes e Genomas de Kyoto) LRR Leucine Rich Repeats (Repetições Ricas em Leucina) LysM Lysin Motif (Motivo de Lisina) MADS-box Box of the Proteins MCM1 from Saccharomyces cerevisiae, AGAMOUS from Arabidopsis thaliana, DEFICIENS from Antirrhinum majus and SRF from Homo sapiens (Conjunto das Proteínas MCM1 de Saccharomyces cerevisiae, AGAMOUS de Arabidopsis thaliana, DEFICIENS de Antirrhinum majus e SRF de Homo sapiens). MEGA Molecular Evolutionary Genetics Analysis (Análises Genéticas e Evolução Molecular) MFS Major Facilitator Superfamily (Superfamília de Facilitadores Principais) MIP Major Intrinsic Protein (Proteína Intrínseca Principal) MS Membrana do Simbiossomo MtAnn Annexin from Medicago truncatula (Anexina de Medicago truncatula) N Nitrogênio NASA National Aeronautics and Space Administration (Agência Espacial Norte Americana) NCBI National Center for Biotechnology Information (Centro Norte Americano dee Biotecnologia e Informação) NFP Nod Factor Perception (Percepção do Fator Nod) NFR Nod Factor Receptor (Receptor de Fator Nod) + NH4 Íon Amônia NIN Nodule Inception Protein (Proteína do Início da Nodulação) NJ Neighbor Joining (Agrupamento por Vizinhança) IX9 - NO3 Íon Nitrato NOD Nodulina NORDEST Rede Nordeste de Biotecnologia NORK Nodulation Receptor Kinase (Receptor Quinase da Nodulação) Nramp Natural resistance-associated macrophage protein (Proteína do Macrófago Associada à Resistência Natural) NSP Nodulation Signaling Pathway (Via de sinalização da Nodulação) ONSA Organization for Nucleotide Sequencing and Analysis (Organização para Sequenciamento e Análise de Nucleotídeos) ORF Open Reading Frame (Quadro Aberto de Leitura) PDB Protein Data Base (Banco de Dados de Proteínas) PIR Protein Information Resources (Recursos de Informações Protéicas) RNA Ribonucleic acid (Ácido Ribonucléico) SAGE Serial Analysis of Gene Expression (Análise Serial da Expressão Gênica) SUCEST Sugarcane EST Project (Projeto EST da Cana-de-açúcar) SYMRK Symbiosis Receptor-Like Kinase (Receptor Quinase da Simbiose) UPGMA Unweighted Pair Group Method with Arithmetic Mean (Método não Polarizado de Agrupamentos aos Pares com Médias Aritméticas) X10 LISTA DE FIGURAS CAPÍTULO 1 Figura 1. Visão geral do ciclo do nitrogênio 20 Figura 2. Representação esquemática da via de transdução de sinal ativado pelos 29 fatores Nod, bem como os tipos e as principais proteínas encontradas nos CAPÍTULO 2 Figura 1: Dendrograms generated after Maximum Parsimony analysis 83 showing relationships among conserved domains in early nodulins (A) ENOD8 and (B) Annexin sequences including Vigna unguiculata orthologs. Figura 2. Dendrograms generated after Maximum Parsimony analysis 84 showing relationships considering conserved domains of late nodulins (A) Sucrose synthase and (B) Glutamine synthase sequences with Vigna unguiculata orthologs. Figura 3. General distribution
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