Identificação e caracterização de duas germinas-like do látex de Calotropis procera

• Ano de defesa: 2017
• Autor(a) principal: Freitas, Deborah Carvalho
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Palavras-chave em Português: Germinas; Látex; Oxalato oxidase
• Link de acesso: http://www.repositorio.ufc.br/handle/riufc/37470

The constant exposure of plants to stress conditions caused by biotic and abiotic factors induces defense mechanisms that promote the production of PR-proteins. In this study, the germin and germin-like proteins (GLPs) belonging to the PR-15 and PR-16 families, respectively, were investigated in latex of C. procera. The transcriptome analysis of this species revealed two sequences with high similarity (greater than 60%) with GLPs, which were named GLCp1 and GLCp2. In silico analysis showed that the GLPs of C. procera consecutively presented 216 and 206 amino acid residues, with apparent molecular weight of 22.79 and 21.79 kDa, values similar to those reported for germin-like. Homology modeling generated reliable models that after undergoing molecular docking studies evidenced a probable oxalate binding site involving three histidine and one glutamate residues. The latex of C. procera was fractionated by chromatographic methods and the seven peaks obtained (P1-P7) were subjected to proteomic analysis. Enzymatic assays detected oxalate oxidase (OxO) activity in the P4 fraction. An electrophoresis of the peaks showed that P4 has proteins of molecular weight between 20-25 kDa, possibly monomers of GLP. Immunoassays using a peptide synthesized from the GLCp1 and GLCp2 sequences were able to recognize P4. The ELISA assay was also performed with latex-free plant material containing seedlings, seedling roots, non-germinated bark-free seeds and non-germinated seeds with and without exposed embryos, in all these latex-free materials there was no recognition by the anti-GLCp-pep antibody, suggesting that these proteins are only found in the latex. The large GLP identity indicates that the two C. procera sequences are germin-like proteins. According to the enzymatic assays they have oxalate oxidase activity, an unusual activity in GLPs, being, therefore, the first report of germin-like proteins with OxO activity in laticifers fluids.