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MAPEAMENTO DOS SÍTIOS DE Dnar 5S E 45S E ORGANIZAÇÃO DA CROMATINA EM REPRESENTANTES DA FAMÍLIA AMARYLLIDACEAE JAUME ST.-HIL

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MAPEAMENTO DOS SÍTIOS DE Dnar 5S E 45S E ORGANIZAÇÃO DA CROMATINA EM REPRESENTANTES DA FAMÍLIA AMARYLLIDACEAE JAUME ST.-HIL EMMANUELLY CALINA XAVIER RODRIGUES DOS SANTOS MAPEAMENTO DOS SÍTIOS DE DNAr 5S E 45S E ORGANIZAÇÃO DA CROMATINA EM REPRESENTANTES DA FAMÍLIA AMARYLLIDACEAE JAUME ST.-HIL. RECIFE-PE 2015 i EMMANUELLY CALINA XAVIER RODRIGUES DOS SANTOS MAPEAMENTO DOS SÍTIOS DE DNAr 5S E 45S E ORGANIZAÇÃO DA CROMATINA EM REPRESENTANTES DA FAMÍLIA AMARYLLIDACEAE JAUME ST.-HIL. Tese apresentada ao Programa de Pós-Graduação em Botânica da Universidade Federal Rural de Pernambuco como parte dos requisitos para obtenção do título de Doutora em Botânica. Orientador: Prof. Dr. Reginaldo de Carvalho Dept° de Genética/Biologia, Área de Genética/UFRPE Co-orientador: Prof. Dr. Leonardo Pessoa Felix Dept° de Fitotecnia, UFPB RECIFE-PE 2015 ii MAPEAMENTO DOS SÍTIOS DE DNAr 5S E 45S E ORGANIZAÇÃO DA CROMATINA EM REPRESENTANTES DA FAMÍLIA AMARYLLIDACEAE JAUME ST.-HIL. Emmanuelly Calina Xavier Rodrigues dos Santos Tese defendida e _________________ pela banca examinadora em ___/___/___ Presidente da Banca/Orientador: ______________________________________________ Dr. Reginaldo de Carvalho (Universidade Federal Rural de Pernambuco – UFRPE) Comissão Examinadora: Membros titulares: ______________________________________________ Dra. Ana Emília de Barros e Silva (Universidade Federal da Paraíba – UFPB) ______________________________________________ Dra. Andrea Pedrosa Harand (Universidade Federal de Pernambuco – UFPE) ______________________________________________ Dr. Felipe Nollet Medeiros de Assis (Universidade Federal da Paraíba – UFPB) ______________________________________________ Dr. Marcelo Guerra (Universidade Federal de Pernambuco – UFPE) Suplentes: ______________________________________________ Dra. Lânia Isis Ferreira Alves (Universidade Federal da Paraíba – UFPB) ______________________________________________ Dra. Sônia Maria Pereira Barreto (Universidade Federal de Pernambuco – UFRPE) iii A minha família, em especial ao meu pai José Geraldo Rodrigues dos Santos que sempre foi o meu maior incentivador e a quem responsabilizo o meu amor pela docência. Dedico. iv AGRADECIMENTOS A Deus, pela iluminação em todos os momentos! Ao Programa de Pós-Graduação em Botânica (PPGB), por permitir o uso de suas dependências. A CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) pela concessão da bolsa de estudo. A coordenadora do programa Profa Dra. Carmen Silvia Zickel, pelo apoio nos momentos que precisei. Meus sinceros agradecimentos! Ao professor Dr. Reginaldo de Carvalho, pela orientação, pelo apoio dispensado e as importantes contribuições durante o desenvolvimento do trabalho. Obrigada por tudo! Ao meu coorientador professor Dr. Leonardo Pessoa Felix, por ter propiciado o meu primeiro contato com a Citogenética vegetal. Não posso deixar de relatar o profundo respeito, carinho e admiração por este ser humano tão incrível. Obrigada pela confiança, profissionalismo e por deixar claro que tenho “cadeira cativa” no citoareia. Os meus mais sinceros agradecimentos! Aos membros da banca examinadora, Dra. Ana Emilia de Barros e Silva, Dra. Andrea Pedrosa Harand, Dr. Felipe Nollet Medeiros de Assis, Dr. Marcelo Guerra, Dra. Lânia Isis Ferreira Alves e Dra. Sônia Maria Pereira Barreto, pelas valiosas contribuições. A minha grande amiga, Dra. Lidiane Feitoza, por ter sido tão essencial na minha formação e por ser um exemplo de ser humano e de profissional. Obrigada, minha querida! Ao meu pequeno grande amigo/irmão, Dr. Felipe Nollet por ser quem é. Pela ajuda profissional e pessoal durante o doutorado. Não tive a sorte de ter um irmão, mas não sabia eu que antes de nascer Deus já o tinha colocado no mundo para preencher esse espaço. Agradeço infinitamente a Citogenética por ter possibilitado esse encontro. Te amo! A minha amiga/irmã/mãe Dra. Lânia Isis, por ter me ensinado a fazer as primeiras lâminas. Como sou sortuda, não?! Agradeço imensamente por sua parceria e amizade sincera. Obrigada por nunca ter me decepcionado e por está comigo sempre, mesmo que a quilômetros de distância. Você está em meu coração. Amo você! A Dra. Ana Emília Barros e Silva por abrir as portas do seu laboratório, assim como o prof. Leonardo Felix, permitindo que eu fique a vontade para voltar para “casa” (Lab Areia-PB). Muito obrigada pelo apoio e conselhos. Ao amigo Erton Medeiros de Almeida que em suas coletas nunca esquece de coletar as “cebolinhas da Manu”. Muito obrigada, querido! Ao meu Amor, Austrique Liano, por ser tão compreensivo, parceiro e carinhoso. Não tenho palavras pra descrever o tamanho da minha gratidão nos momentos que mais precisei. Te amo! Aos meus pais José Geraldo Rodrigues dos Santos e Célia Maria Xavier dos Santos, e as minhas irmãs Gabriele Xavier e Camila Xavier pelo suporte, orações e momentos felizes. v Em especial ao meu pai, meu maior exemplo. Por me levar ainda criança em seus experimentos de campo durante seu Doutorado. Obrigada por mostrar que a educação é libertadora. Obrigada por ser um exemplo de superação não só para mim, mas também para seus alunos. Quero e sempre quis ser como o meu pai, espero um dia conseguir chegar perto. Aos meus tios, tias e primas professores que desde sempre despertaram em mim o amor pela leitura e que mesmo diante as dificuldades conseguem mudar vidas. Vocês me inspiram! A minha avó, também professora, Antonia Xavier, o ser humano mais nobre e lúcido que conheço. Obrigada por ser a avó dos sonhos de qualquer um e por ter me ensinado tantas lições valiosas. Te amo! Aos meus colegas de laboratório, tanto do citoareia como do citorural, pela troca de ideias, pelos bons momentos vivenciados e pelas valiosas lições. A todos os amigos da graduação, mestrado, doutorado e da vida pelos maravilhosos momentos vivenciados: aulas, viagens, risadas, conversas e festas. A todos os funcionários da UFRPE e UFPB, em especial a Kênia Muniz e a Luciana Ledra, pelo apoio nos momentos que precisei. Por fim, agradeço a todos que contribuíram de alguma forma, ou à sua forma, para a realização deste trabalho, meus sinceros agradecimentos. vi LISTA DE FIGURAS FUNDAMENTAÇÃO TEÓRICA Pág. Figura 1 Distribuição geográfica da família Amaryllidaceae. Subfamília 28 Agapanthoideae (a); Subfamília Amaryllidoideae (b); Subfamília Allioideae: tribo Allieae (c), tribo Tulbaghieae (d), tribo Gilliesieae (e). Figura 2 Principais etapas da técnica de hibridização in situ fluorescente. A 36 marcação da sonda (a) realizada independentemente da preparação da lâmina (b). Posteriormente, a sonda e o DNA cromossômico são desnaturados (c, d) e, quando colocados em contato, a sonda hibridiza in situ (e) com o DNA-alvo. A marcação da sonda pode ser detectada por anticorpos ligados a fluorocromos (f) e visualizada diferencialmente ao microscópio (g). Figura 3 Diagrama esquemático da estrutura das histonas. (a) Proteínas dos 39 nucleossomos são designadas: H2A (histona 2A), H2B (histona 2B), H3 (histona 3) e H4 (histona 4). Cada histona está presente em duas cópias, de modo que o DNA (preto) envolve um octâmero de histonas. (b) As caudas amino-terminais de histonas do núcleo. Lisinas (K) nas caudas amino-terminais das histonas H2A, H2B, H3 e H4 são potenciais locais de acetilação/desacetilação para a histona acetiltransferases (HAT) e histonas deacetilases (HDAC). A, acetil; C, carboxila; E, ácido glutâmico; M, metil; N, amino-terminal; P, fosfato; S, serina; Ub, ubiquitina. CAPÍTULO I: Localização cromossômica dos sítios de DNAr e heterocromatina, e suas implicações para a evolução cariotípica em representantes dos gêneros Habranthus, Hippeastrum e Zephyranthes (subtribo Hippeastrinae: Amaryllidaceae) Figura 1 Metáfases mitótica de espécies do gênero Hippeastrum (2n = 22) coradas 190 com CMA (amarelo), DAPI (azul) e hibridizadas com sondas de DNAr 5S (vermelho) e 45S (verde). (a-b) Hippeastrum glaucescens; (c-d) H. puniceum; (e-f); H. striatum (g-h) H. stylosum; (i-j) Hippeastrum sp1. Insertos mostram maior ampliação em a, c, e, g, i das bandas CMA+/DAPI- e em b, d, f, h, j dos sítios de DNAr 5S e 45S. Barra em j corresponde a 10µm. Figura 2 Cariogramas das espécies do gênero Hippeastrum coradas com DAPI (azul) e hibridizadas in situ com sondas de DNAr 5S (vermelho) e 45S (verde). (a) Hippeastrum glaucescens; (b) H. puniceum; (c); H. striatum; (d) H. stylosum; (e) Hippeastrum sp1. Todos os cariogramas foram organizados por ordem decrescente de tamanho. A barra em e vii Pág. corresponde a 10 μm. A linha pontilhada indica a posição do 191 centrômero. Figura 3 Células metafásicas de espécies dos gêneros Habranthus e Zephyranthes 192 coradas com CMA (amarelo), DAPI (azul) e hibridizadas com sondas de DNAr 5S (vermelho) e 45S (verde). (a-b) Habranthus brachyandrus (2n = 12); (c-d) H. sylvaticus (2n = 12); (e-f) Zephyranthes flavissima (2n = 14); (g-h) Z. mesochloa (2n = 24); (i-j) Zephyranthes aff. stellaris (2n = 20). Insertos mostram maior ampliação em a, c, e, g, i das bandas CMA+/DAPI- e em b, d, f, h, j dos sítios de DNAr 5S e 45S. Setas em b apontam bandas mais fortemente coradas com DAPI após a FISH (inserto). Cabeça de setas em i indicam bandas CMA+. Barra em j corresponde a 10µm. Figura 4 Cariogramas das espécies do gênero Habranthus e Zephyranthes coradas 193 com CMA (amarelo) e DAPI (azul) e hibridizadas in situ com sondas de DNAr 5S (vermelho) e 45S (verde). (a) Habranthus brachyandrus; (b) H. sylvaticus (c) Zephyranthes flavissima; (d) Z. mesochloa; (e) Zephyranthes aff. stellaris. Todos os cariogramas foram organizados por ordem decrescente de tamanho. A barra em e corresponde a 10 μm. A linha pontilhada indica a posição do centrômero. CAPÍTULO II:
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