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Isolamento E Caracterização in Silico De Ciclotídeos Em Milho (Zea Mays) E Centeio (Secale Cereale)

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Isolamento E Caracterização in Silico De Ciclotídeos Em Milho (Zea Mays) E Centeio (Secale Cereale) UNIVERSIDADE FEDERAL DE PERNAMBUCO CENTRO DE CIÊNCIAS BIOLÓGICAS PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS DISSERTAÇÃO DE MESTRADO SHEYLA CARLA BARBOSA DA SILVA LIMA Isolamento e caracterização in silico de ciclotídeos em milho (Zea mays) e centeio (Secale cereale) Recife 2015 i SHEYLA CARLA BARBOSA DA SILVA LIMA Isolamento e caracterização in silico de ciclotídeos em milho (Zea mays) e centeio (Secale cereale) Dissertação apresentada ao programa de Pós Graduação em Ciências Biológicas da Universidade Federal de Pernambuco, como requisito final exigido para a obtenção do título de Mestre em Ciências Biológicas, área de concentração: Biotecnologia. Orientadora: Profa Drª Valesca Pandolfi Coorientadora: Profª Drª Ana Maria Benko Iseppon Recife 2015 Catalogação na fonte Elaine Barroso CRB 1728 Lima, Sheyla Carla Barbosa da Silva Isolamento e caracterização in silico de ciclotídeos em milho (Zea mays) e centeio (Secale cereale)/ Sheyla Carla Barbosa da Silva Lima– Recife: O Autor, 2015. 149 folhas: il., fig., tab. Orientadora: Valesca Pandolfi Coorientadora: Ana Maria Benko Iseppon Dissertação (mestrado) – Universidade Federal de Pernambuco. Centro de Ciências Biológicas. Biotecnologia, 2015. Inclui bibliografia e anexos 1. Bioinformática 2. Peptídeos 3. Gramínea I. Pandolfi, Valesca (orientadora) II. Iseppon, Ana Maria Benko (coorientador) III. Título 660.6 CDD (22.ed.) UFPE/CCB-2015-203 ii Isolamento e caracterização in silico de ciclotídeos em milho (Zea mays) e centeio (Secale cereale) Dissertação apresentada ao programa de Pós Graduação em Ciências Biológicas da Universidade Federal de Pernambuco, como requisito final exigido para a obtenção do título de Mestre em Ciências Biológicas, área de concentração: Biotecnologia. Data de Aprovação: 24/02/2015 COMISSÃO EXAMINADORA ____________________________________________ Profa. Drª Valesca Pandolfi (Orientadora - Universidade Federal de Pernambuco) ____________________________________________ Profa. Drª. Ana Maria Benko-Iseppon (Membro interno - Universidade Federal de Pernambuco) ____________________________________________ Dr. José Ribamar Costa Ferreira Neto (Membro externo - Universidade Federal de Pernambuco) Recife 2015 iii À minha família, por todo amor, dedicação e apoio. Dedico. iv AGRADECIMENTOS Primeiramente, agradeço a Deus pelo dom da vida e por me proporcionar mais uma conquista, apesar de tantos obstáculos Ele está ao meu lado guiando-me e dando- me força. Aos meus pais, Lucilia e Robson que estiveram comigo desde a primeira vez da escrita do meu nome e não mediram esforços para que hoje estivesse onde estou. Mostraram o caminho difícil, mas recompensador dos estudos. Ao meu irmão que amo tanto pelos momentos únicos que me proporcionou e por todo apoio. Ao meu esposo Cassiano Neto pelos anos de dedicação, amor, apoio e em especial compreensão nos momentos mais difíceis desta jornada. Obrigada meu amor, tudo que tenho conquistado também devo a você. À minha orientadora Drª Valesca Pandolfi, pela oportunidade de ser orientanda, por toda ajuda e por acreditar na minha capacidade. À minha coorientadora e chefe do Laboratório de Genética e Biotecnologia Vegetal (LGBV) Drª Ana Maria Benko-Iseppon, pela oportunidade de integrar a equipe deste laboratório, pela confiança e ajuda. Ao meu coorientador não documentado M.Sc. João Pacífico por todo conhecimento passado e construído de forma paciente e didática. Por me ajudar a desvendar os “mistérios” da bioinformática, por toda ajuda no desenvolvimento deste trabalho, além dos momentos de risadas e pela carona de quase todos os dias. Aos amigos que encontrei neste caminho: Stephani Soares e Cecília Bernardo pelas conversas e companhia nos primeiros momentos dentro do LGBV; à Karla Kamila por ser tão solícita em transmitir conhecimentos e pelas risadas que tivemos; aos meninos da “bioinfo” Marx, Pedro e Rômulo por toda ajuda; à Rebeca Rivas e Ana Rafaela pela participação especial. A Bruna, Flávia, Hévila e Silvany pelos momentos incríveis que passamos juntas no laboratório e fora dele, pelas risadas e brincadeiras que tornaram meus dias mais leves e os congressos mais animados. Pelas diversas conversas a respeito do futuro e tantos outros assuntos. Obrigada pela amizade! v A todos que fizeram e fazem parte do LGBV: Polyana, Mitaly, Lidiane, Anne, Manu, Santelmo, Diego, Neto, Wilson, Ivamberto, Jaiza, pelos momentos de crescimento profissional e momentos de intensa alegria. À Vanessa pela ajuda na resolução de problemas e pelas palavras mágicas “vai dar tempo” nos momentos de desespero. À Universidade Federal de Pernambuco e Pós–Graduação em Ciências Biológicas, por todo suporte neste período e à Adenilda que tenta nos ajudar do jeito dela (risos). Aos familiares pelo apoio e incentivo, primos, primas, tias, tios, minhas madrinhas Ione e Lucineide. Ao meu sogro Cassiano Filho pelas palavras de incentivo e minha sogra Girleide que direta ou indiretamente contribuíram para essa conquista. Às minhas amigas desde o ensino médio Anne, Izabel, Vera, Fernanda e Andreza que mesmo distantes, se preocuparam, me apoiaram e sempre torcem por mim, obrigada pela amizade. Aos professores que compartilharam o conhecimento e suas experiências. Ao suporte financeiro concedido pelas agências de fomento à pesquisa, CAPES (Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) e FACEPE (Fundação de Amparo à Ciência e Tecnologia de Pernambuco). Enfim, a todos que de alguma maneira direta ou indiretamente contribuíram para a realização deste trabalho. Muito obrigada! vi “Ó minha alma se alegra somente no Senhor, porque d’Ele vem a minha esperança. ” Salmos 62-5 vii RESUMO Ciclotídeos são uma classe de peptídeos antimicrobianos (AMPs - do inglês Antimicrobial peptide) cíclicos de plantas, compostos de, aproximadamente, 30 resíduos de aminoácidos, sendo seis cisteínas conservadas e conectadas por três pontes de dissulfeto. Sua expressão é constitutiva, tendo sua principal função na defesa vegetal contra patógenos, que podem causar perdas significativas em culturas importantes para a agricultura, como no caso da família Poaceae que apresenta destacada importância econômica no Brasil e no mundo. Nesse estudo foi conduzida uma busca por genes relacionados a ciclotídeos vegetais, disponíveis em bancos de dados de acesso restrito e público, com vistas ao isolamento e caracterização in silico desses peptídeos. Através da busca nos genomas de Hevea brasiliensis, Manihot esculenta, Ricinus communis, Sorghum bicolor e Zea mays; bem como no transcriptoma de Vigna unguiculata foi verificado que apenas o genoma de Zea mays apresentou dois possíveis genes codificadores de ciclotídeos. Assim, primers foram desenhados para o isolamento destes genes em milho. Além da espécie Z. mays, as espécies Triticum aestivum (trigo) e Secale cereale (centeio), foram utilizadas para a tentativa de isolamento a partir dos pares de primers desenhados. Foram obtidos 19 fragmentos (amplicons), sendo quatro deles (zm315, zm316, zm317, sc359) com o domínio ciclotídeo, os três primeiros de milho e o último de centeio. Essas quatro sequências foram, então, submetidas a uma caracterização in silico, para predição da estrutura secundaria, terciaria e função predita. Verificou-se que esses peptídeos apresentam as seis cisteínas conservadas, três pontes dissulfeto e o padrão de aminoácidos entre as cisteínas, similar aos encontrados em ciclotídeos. Ainda foi possível a predição de algumas propriedades físico-químicas e modelagem por homologia para as quatro proteínas, o que mostrou a qualidade e confiabilidade dos modelos. Sugere-se que dois dos ciclotídeos isolados (zm315, zm316) pertençam a uma nova classe de peptídeos lineares, mas com características de ciclotídeos. Palavras-Chave: Bioinformática, Modelagem por homologia, AMPs, Poaceae. viii ABSTRACT Cyclotides are a class of cyclic antimicrobial peptides (AMPs) present on plants, composed by approximately 30 amino acid residues, including six conserved cysteines connected by three disulphide bridges. Its expression is constitutive, with main function on plant defense against pathogens, that may cause significant losses in important cultivars, as in the case of Poaceae, a family that presents economic importance for the agriculture in Brazil and worldwide. This study performed a search for genes related to plant cyclotides, available in restricted and public access databases, aimed at their in silico isolation and characterization. Searching for these peptides in Hevea brasiliensis, Manihot esculenta, Ricinus communis, Sorghum bicolor, Vigna unguiculata and Zea mays genomes, we obtained two possible genes encoding Cyclotides in Z. mays. Thus, primers were designed for the isolation of these genes in maize as well in wheat (Triticum aestivum) and rye (Secale cereale) species. We obtained 19 amplicons and four of them (zm315, zm316, zm317, sc359) presented cyclotide domain. These four sequences were then subjected to in silico characterization, for predicting their secondary and tertiary structures, as well their function. It was found that these peptides present six conserved cysteines, three disulphide bridges and the amino acid pattern between the cysteines similar to those found in cyclotides. It was also possible to predict some physical chemical properties and also building a 3D protein by homology modeling for the four peptides, presenting high quality and reliability. Our analysis indicates that two isolated cyclotides (zm315, zm316) appear to
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