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Estudo Da Diversidade E Atividade Bacteriana Em Solos De Floresta E Sob Cultivo De Cana-De-Açúcar

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Estudo Da Diversidade E Atividade Bacteriana Em Solos De Floresta E Sob Cultivo De Cana-De-Açúcar UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL ESTUDO DA DIVERSIDADE E ATIVIDADE BACTERIANA EM SOLOS DE FLORESTA E SOB CULTIVO DE CANA-DE-AÇÚCAR Wellington Marcelo Queixas Moreira Biólogo 2013 UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL ESTUDO DA DIVERSIDADE E ATIVIDADE BACTERIANA EM SOLOS DE FLORESTA E SOB CULTIVO DE CANA-DE-AÇÚCAR Wellington Marcelo Queixas Moreira Orientadora: Prof. Dra.Lúcia Maria Carareto Alves Tese apresentada à Faculdade de Ciências Agrárias e Veterinárias – UNESP, Câmpus de Jaboticabal, como parte das exigências para a obtenção do título de Doutor em Microbiologia Agropecuária. 2013 Moreira, Wellington Marcelo Queixas M838e Estudo da diversidade e atividade bacteriana em solos de floresta e sob cultivo de cana-de-açúcar / Wellington Marcelo Queixas Moreira. – – Jaboticabal, 2013 xiii, 103 p.: il.; 28 cm Tese (doutorado) - Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, 2013 Orientadora:Lúcia Maria Carareto Alves Banca examinadora: Antonio Carlos Monteiro, Haroldo Alves Pereira Júnior, Luciano Takeshi Kishi, Mariana Carina Frigieri Salaro Bibliografia 1. Ciclos biogeoquímicos. 2. gene 16S rRNA. 3. Saccharum spp. 4. Hipervariáveis V I. Título. II. Jaboticabal-Faculdade de Ciências Agrárias e Veterinárias. CDU 579.22:633.61 Ficha catalográfica elaborada pela Seção Técnica de Aquisição e Tratamento da Informação – Serviço Técnico de Biblioteca e Documentação - UNESP, Câmpus de Jaboticabal. DADOS CURRICULARES DO AUTOR Wellington Marcelo Queixas Moreira – nascido em Bebedouro (SP), em 24 de fevereiro de 1983, graduou-se em Ciências Biológicas pelo Centro Universitário UNIFAFIBE em Bebedouro (SP), no ano de 2004. Ingressou no Curso de Especialização, em 2006, recebendo o título de Especialista em Biologia Molecular e Biotecnologia, pela Universidade de Franca (UNIFRAN) em junho de 2007. Em agosto de 2007, iniciou o Mestrado, no curso de Pós-graduação em Microbiologia, Área de concentração em Microbiologia Agropecuária, na Faculdade de Ciências Agrárias e Veterinárias (FCAV) da Universidade Estadual Paulista (UNESP), Jaboticabal (SP), recebendo o título de mestre em julho de 2009. Em agosto do mesmo ano, iniciou o curso de Doutorado também no Programa de Microbiologia Agropecuária. “No meio da confusão, encontre a simplicidade. A partir da discórdia, encontre a harmonia. No meio da dificuldade reside a oportunidade”. Albert Einstein Aos meus pais João e Marli, por toda dedicação, educação e confiança. À Gabriela, sempre companheira. E a meus familiares e amigos Dedico com carinhocarinho.... AGRADECIMENTOS Agradeço a DEUS e a minha família À Profa. Dra. Lúcia Maria Carareto Alves, pela orientação, amizade, paciência e confiança depositada. À Profa. Dra. Eliana Gertrudes de Macedo Lemos, por todo suporte, idéias e incentivo no decorrer deste trabalho. Ao Dr. João Carlos Campanharo pelos constantes auxílios e amizade. Aos professores doutores: Antônio Carlos Monteiro, Eliana Gertrudes de Macedo Lemos, Haroldo Alves Pereira Júnior, Luciano Takeshi Kishi, Maria Inês Tiraboschi Ferro, Mariana Carina Frigieri Salaro, Silvana Silvana Pompéia do Val- Moraes e Renata Aparecida de Andrade pelas sugestões e correções no exame de qualificação e defesa. À amiga Dra. Silvana Pompéia do Val-Moraes pela amizade e colaboração no decorrer deste e de outros trabalhos. À Gabriela Sanches Perez pelo apoio, carinho, paciência e companheirismo incondicional. Ao Dr. Luciano Takeshi Kishi por todo suporte no decorrer deste. A todos os companheiros do Laboratório de Bioquímica de Microrganismos e Plantas pela amizade e por tudo mais. A todos os amigos do Centro Universitário Unifafibe pela amizade e companheirismo. Ao professor Ms. Hélio José dos Santos Souza e prof. Dr. Rinaldo Guariglia do Centro Universitário Unifafibe, meus sinceros agradecimentos. E o meu obrigado a todos que direta ou indiretamente sempre contribuíram para realização deste trabalho. Muito Obrigado. v SUMÁRIO Página RESUMO ... ...................................................................................................... .......x SUMMARY ........................................................................................................... xii 1. INTRODUÇÃO .................................................................................................. 1 2. REVISÃO DE LITERATURA ............................................................................ 3 2.1. Microrganismos e os ciclos biogeoquímicos ................................................. 3 2.2. Vegetação do Estado de São Paulo .............................................................. 7 2.3. Cultura da cana-de-açúcar .......................................................................... 10 2.4. Estudos de microrganismos independente de técnicas de cultivo ............. 12 2.5. Sequenciamento de alta performance .................................................... 14 3. MATERIAL E MÉTODOS ................................................................................ 18 Parte I – Área de estudo e amostragem do solo, obtenção e análise do DNA metagenômico .............................................................................................. 18 1. Áreas de estudo ...................................................................................... 18 1.1. Área 1: Santa Rita do Passa Quatro ............................................... 18 1.2. Área 2: Mococa ............................................................................... 19 2. Coleta do solo ......................................................................................... 20 3. Extração do DNA metagenômico do solo ................................................ 22 4. Análise da integridade e quantificação do DNA metagenômico .............. 22 Parte II – Construção das bibliotecas dos genes 16S rRNA do DNA metagenômico das amostras de solo .......................................................... 22 1. Amplificação por PCR da região 16S rDNA ............................................. 22 2. Eluição e purificação dos produtos da PCR ............................................ 23 3. Clonagem em vetor pGEM ® T Easy ......................................................... 24 4. Transformação das células competentes ................................................. 25 5. PCR para sequenciamento ..................................................................... 25 6. Análise das sequencias ............................................................................ 26 Parte III – Amplificação da região Vi (V1-V2) do 16S rDNA do DNA metagenômico do solo para análise da diversidade bacteriana baseada em Sequenciamento de Nova Geração (NGS) ................................................... 26 1. Amplificação por PCR da região Vi do 16S rDNA ..................................... 27 2. Eluição e purificação dos fragmentos de PCR da região Vi do 16S ......... 28 3. Quantificação das amostras (Qubit ®) ........................................................ 29 vi 4. Construção e validação das bibliotecas .................................................... 28 5. Normalização das amostras ........................................................................ 29 6. Clusterização e sequenciamento ................................................................. 30 7. Análise de Dados ......................................................................................... 31 4. RESULTADOS E DISCUSSÃO ................................................................... 32 Parte I: Análise das características do solo e extração do DNA metagenômico do solo .................................................................................. 32 1. Análise das características físico-químicas das diferentes amostras de solo 32 2. Análise do DNA metagenômico .................................................................. 34 PARTE II: Bibliotecas de fragmentos 16S rDNA do DNA metagenômico do solo ................................................................................................................ 37 1. Amplificação e clonagem dos fragmentos 16S rDNA ................................ 37 2. Clonagem e validação das bibliotecas 16S rDNA ..................................... 38 3. Análise das bibliotecas de fragmentos 16S rDNA do DNA metagenômico do solo .......................................................................................................... 39 3.1. Análise da diversidade bacteriana nos solos ......................................... 39 3.1.1 Análise individual no banco NCBI ..................................................... 39 3.1.2. Comparação das sequencias de diferentes bibliotecas no RDP II .. 43 3.2. Análise da atividade microbiana nos solos em relação aos ciclos biogeoquímicos ............................................................................................. 47 PARTE III: Análise da diversidade bacteriana baseada em sequenciamento de nova geração (NGS) da região Vi do gene 16S rRNA do DNA metagenômico do solo .................................................................................. 55 1. Amplificação dos Fragmentos Vi do 16S rDNA ......................................... 55 2. Validação das bibliotecas .......................................................................... 57 3. Análise das comunidades bacterianas dos solos baseadas em unidades taxonômicas operacionais (OTUs) ................................................................ 58 4. Análise da diversidade bacteriana nos solos
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