Prospecção de peptídeos antimicrobianos de soja (Glycine max) utilizando ferramentas proteômicas

• Autor(a) principal: Barbosa, Meire de Oliveira
• Data de Publicação: 2008
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/287
• topic: Proteômica; Peptídeos antimicrobianos; Soja; Defesa de plantas; Proteomics; Antimicrobial peptides; Soybean; Defense processes in plants; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS::PROTEINAS

The techniques based on the separation by two-dimensional liquid chromatography (TDLC) or multi-dimensional (MDLC), coupled with mass spectrometry analysis, have been used in separation of proteins and peptides for bioprospection of antimicrobial activities. This work aimed to bioprospect antimicrobial peptides (AMPs) of soybean (Glycine max) by using proteomics tools. Peptides from non- germinated (NGSS) and germinated for 48 hours (GSS48) soybean seeds were extracted and fractionated by salt and heating, followed by dialysis, anion-exchange (AEC) and reversed phase (C18 RPHPLC) chromatographic steps, and mass spectrometry analysis (MS). The comparisons of the chromatographic profiles (AEC and RP-HPLC) from NGSS and GSS48 suggested that different peptides are synthesized, showing molecular weight (MW) lower than 10 kDa by MS analysis. A biorrevelation-assay that used the cationic fraction obtained from SGS48 showed that compounds with MW lower than 14-kDa were able to inhibit the bacterium Ralstonia solanacearum growth. Thus, to improve knowledge about GSS48AMPS´s characteristics, different techniques for clearance and purification of the extracts were evaluated, and a new protocol to bioprospect the peptides was proposed, composed by the following steps: extraction, ammonium sulfate fractionation (35-75% sat.), ultrafiltration (membrane of 30 and 1 kDa cut-off) and TDLC (molecular exclusion chromatography - MEC and C18 RP- HPLC). The fractions obtained after the C18 RP-HPLC were heated by two hours for solvent removal, and the recovered fractions were used for antimicrobial assays, SDS-Tricine- PAGE and MS analysis. Various fractions, obtained after the C18 RP-HPLC after heating were active against five plant-pathogens. Molecules with MW below 10-kDa were present in these fractions The comparison of the differential synthesis of AMPs in NGSS and in GSS48 showed that the germinated seeds present a potential for the identification of peptides with great functional importance, that are probably involved in the defense processes in plants. The adjustment of this protocol allowed an enrichment of AMPs in the SSG48 samples, which are resistant to high temperature and to the contact with acetonitrile and TFA, that were not yet described in the literature. These characteristics could be exploited for biotechnological application in agribusiness. Simultaneously, by using bioinformatic tools, searches were conducted in the database NCBI in order to identify DNA sequences coding for AMPS in soybean. A sequence of a putative defensin was recovered and used as a template for cloning in Escherichia coli. The obtained plasmid of expression (pPIC 9) was linearized and used for the transformation of Pichia pastoris. A His+ (P. pastoris) transformed colony was used for evaluation of the defensin expression and for SDS-Tricine-PAGE, in order to check their expression. The assessment by SDS-Tricine-PAGE failed to confirm the expression of defensin so far, under the conditions used. The identification of a putative defensin and the cloning procedure were carried-out successfully, and its expression is being evaluated aiming to develop various characterizations, which probably will provide different biotechnological applications for the peptides.