Identificação de peptídeos antimicrobianos de folhas de berinjela para o controle de patógenos de plantas

• Autor(a) principal: Almeida, Hebréia Oliveira
• Data de Publicação: 2007
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2476
• topic: Peptídeos antimicrobianos; Defesa de plantas; Berinjela; Antimicrobial peptides; Plant defense; Eggplant; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS::PROTEINAS

The antimicrobial peptides (AMPs) are produced by animals, plants, insects and others organisms as an efficient, flexible and economical strategy of defense, consuming low energy and biomass, since they are small molecules. In plants, ten families of AMPs were identified, that are involved in the constitutive barrier of the plant defense, with molecular mass from 2 to 9 kDa. Based on the known importance of the AMPs as possible active molecules to be used for the development of defense agents for pharmaceutical applications or to the development of new classes of agriculture defensives, the goal of this project was to detect, extract and purify AMPs forms, that are present in young plants and in fully-expanded leaves of eggplants, and to evaluate the action of these peptides in vitro e in vivo against plant pathogens with commercial interest. Afterthat, this work aims the cloning and the heterolog expression in order to characterize the molecular structure of the AMPs. This project also have the goal to prospect the antimicrobial potencial of parcial-purified extracts from young plants and from fully-expanded leaves of eggplant, aiming possible biotechnological applications to control plant-pathogens growth. Leaves from 60-days plants and young plants (5 or 15 cm) of eggplants were triturated and macerated (Tris-HCl buffer, EDTA, PMSF, benzamidine and thiourea), the extract was centrifuged and the supernatant corresponded to the Soluble Extract (SE). The precipitate was washed (water), extracted with LiCl (EDTA, PMSF, benzamidina and thiourea), the homogenate was centrifuged and the supernatant corresponded to the Cell-Wall Extract (CWE). SE e CWE from the three development states were fractionated (ammonium sulfate and heating), desalted, lyophilized and separated by anionic-exchange chromatography (DEAE-Sepharose-FPLC). They were obtained two cationic (CP1 e CP2) and one anionic (AP) peaks to each evaluated-stage, that were submitted to growth-inhibition tests of the two plant pathogenic bacteria Ralstonia solanacearum and Clavibacter michiganensis subsp. michiganensis growth. The results indicated a best inhibition rate by CWE of young plants (5 cm), while the SE from mature plants (15 cm and leaf) was more effective. SE from expanded-leaves were submitted to an anionic-exchange chromatography in Q-Sepharose-FPLC and the five obtained pools were submitted to antibacterial tests, and PC1 was submitted to an inativation- or death-tests of plant nematodes. PC1 were selected as a promise to AMPs bioprospection, because it shown an antimicrobial effect against the two evaluated bacteria, and a nematicide effect against the nematode Meloidogyne incognita, and it was then separated by a C18-RP-HPLC. From all collected peaks, twelve were analyzed by mass spectrometry and two of them (peaks 18 and 24) presented peptides with mass from 2 and 9 kDa. The remaining-ES and -EP of expanded-leaves were separated in a Sephadex G-10 column. They were obtained four pools for each ES- and EP-separations, that were submitted to antimicrobial tests in vitro against the two plant pathogenic bacteria above mentioned, and in vivo against Ralstonia solanacearum infection in tomato plant. Some pools showed as a promise, especially the ES-pool 1 obtained by the separation in the Sephadex G-10 column, from that two fractions were separated in a C18-RP-HPLC column, and the recovered peaks were submitted to develop mass spectrometry analysis. They were found molecules with molecular mass in the peptide range. The amino acidic sequences of the peptides present in the fractions corresponding to the peaks 9 and 14 were determinated, and they need to be confirmed. The others peptide peaks will be submitted to mass spectrometry analysis,amino acidic sequenting, and antimicrobial tests, aiming to select effective peptides against plant pathogens that are of interest in the agribusiness.