Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-06-10T19:08:01Z No. of bitstreams: 1 ArianeFerreiraLacerda_TESE.pdf: 2669795 bytes, checksum: 86dd6a13d7f026cc0544d3cb0e76e5f1 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-06-13T20:26:33Z (GMT) No. of bitstreams: 1 ArianeFerreiraLacerda_TESE.pdf: 2669795 bytes, checksum: 86dd6a13d7f026cc0544d3cb0e76e5f1 (MD5) / Made available in DSpace on 2016-06-13T20:26:34Z (GMT). No. of bitstreams: 1 ArianeFerreiraLacerda_TESE.pdf: 2669795 bytes, checksum: 86dd6a13d7f026cc0544d3cb0e76e5f1 (MD5) Previous issue date: 2015-06-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A inefic?cia de pesticidas qu?micos no controle de fungos fitopatog?nicos na agricultura e a frequente incid?ncia de doen?as humanas causadas por bact?rias resistentes a antibi?ticos levam ? busca por compostos antimicrobianos alternativos. Neste contexto, defensinas vegetais constituem uma promissora ferramenta no controle de agentes patog?nicos tanto de plantas quanto de humanos. Defensinas de plantas s?o pept?deos cati?nicos com aproximadamente 50 res?duos de amino?cidos, ricas em ciste?nas e cuja estrutura tridimensional ? bastante conservada entre as diferentes esp?cies vegetais. Estas mol?culas de a??o antimicrobiana representam um importante componente inato da resposta de defesa vegetal contra pat?genos e s?o expressas em diversos tecidos da planta, como folhas, tub?rculos, flores, vagens e sementes. O presente trabalho teve por finalidade a avalia??o da atividade antimicrobiana de duas defensinas vegetais contra diferentes esp?cies de fungos fitopatog?nicos e bact?rias patog?nicas ao homem. A defensina Drr230a, cujo gene foi isolado de ervilha (Pisum sativum) e a defensina CD1, cujo gene foi identificado no transcriptoma de caf? (Coffea arabica) foram subclonadas em vetor de express?o de levedura e expressas em Pichia pastoris. O gene cd1 foi subclonado em duas formas recombinantes: CD1tC, contendo uma sequ?ncia codificante para seis histidinas (6xHis) na regi?o C-terminal do pept?deo e CD1tN, contendo sequ?ncia codificante para 6xHis na regi?o N-terminal. No caso da defensina Drr230a, a sequ?ncia codificante para 6xHis foi inserida apenas na regi?o N-terminal da prote?na. Ensaios de atividade antimicrobiana das prote?nas recombinantes purificadas rDrr230a e rCD1 contra Phakopsora pachyrhizi, agente causal da ferrugem asi?tica da soja, foram realizados para analisar a inibi??o da germina??o de esporos in vitro e a severidade da doen?a causada pelo fungo in planta. As duas defensinas recombinantes testadas foram capazes de inibir a germina??o de uredosporos de P. pachyrhizi, n?o havendo diferen?a entre a a??o antimicrobiana de CD1tC e CD1tN. Ademais, rDrr230a e rCD1 reduziram drasticamente a severidade da ferrugem asi?tica da soja, conforme demonstrado em ensaios in planta. Apesar de rCD1 n?o ter sido capaz de inibir a prolifera??o das bact?rias patog?nicas humanas Staplylococcus aureus e Klebsiella pneumoniae, rCD1 mostrou-se capaz de inibir o crescimento do fungo fitopatog?nico Fusarium tucumaniae, causador da s?ndrome da morte s?bita da soja. Os resultados obtidos mostram que tais defensinas vegetais s?o candidatas ?teis para serem utilizadas em programas de engenharia gen?tica de plantas para controlar doen?as f?ngicas impactantes na agricultura. / The inefficiency of chemical pesticides to control phytopathogenic fungi in agriculture and the frequent incidence of human diseases caused by bacteria which are resistant to antibiotics lead to the search for alternative antimicrobial compounds. In this context, plant defensins are a promising tool for the control of both plant and human pathogenic agents. Plant defensins are cationic peptides of about 50 amino acid residues, rich in cysteine and whose tridimensional structure is considerably conserved among different plant species. These antimicrobial molecules represent an important innate component from plant defense response against pathogens and are expressed in various plant tissues, such as leaves, tubers, flowers, pods and seeds. The present work aimed at the evaluation of the antimicrobial activity of two plant defensins against different phytopathogenic fungi and pathogenic bacteria to humans. The defensin Drr230a, whose gene was isolated from pea (Pisum sativum), and the defensin CD1,whose gene was identified within coffee (Coffea arabica) transcriptome, were subcloned in yeast expression vector and expressed in Pichia pastoris. The gene cd1 was subcloned as two different recombinant forms: CD1tC, containing a six-histidine sequence (6xHis) at the peptide C-terminal region and CD1tN, containing 6xHis coding sequence at the N-terminal region. In the case of the defensin Drr230a, the 6xHis coding sequence was inserted only at the N-terminal region. Assays of the antimicrobial activity of the purified recombinant proteins rDrr230a and rCD1 against Phakopsora pachyrhizi, causal agent of soybean Asian rust, were performed to analyze the in vitro spore germination inhibition and disease severity caused by the fungus in planta. Both recombinant defensins were able to inhibit P. pachyrhizi uredospore germination, with no difference between the antimicrobial action of either CD1tC or CD1tN. Moreover, rDrr230a and rCD1 drastically reduced severity of soybean Asian rust, as demonstrated by in planta assays. In spite of the fact that rCD1 was not able to inhibit proliferation of the human pathogenic bacteria Staplylococcus aureus and Klebsiella pneumoniae, rCD1 was able to inhibit growth of the phytopathogenic fungus Fusarium tucumaniae, that causes soybean sudden death syndrome. The obtained results show that these plant defensins are useful candidates to be used in plant genetic engineering programs to control agriculture impacting fungal diseases.