Análise da expressão de enterolobina em sementes e calos vegetais de Enterolobium contortisiliquum

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

Cytolytic toxins are produced by a variety of living organisms, particularly bacteria, certain insects, poisonous reptiles and stinging marine invertebrates. Many of these toxins appear to function simply by forming pores in cell membranes, disrupting the permeability barrier and leading eventually to cell death. However, few examples have been studied in plants. Enterolobin was the first plant cytolytic protein described in the literature. It is a membrane pore-forming protein extracted from Enterolobium contortisiliquum seeds. The current work aimed at assaying E. contortisiliquum callus as possible source material for enterolobin purification, and comparing protein expression in callus and seeds of E. contortisiliquum. For callus production, seeds were randomly selected and scarified, and then overnight incubated in distilled water. A solution of 30 % sodium hipochloride was used for seed decontamination. Each seed was placed into a tube containing 20 mL of MS-61 medium. Seed germination and development was allowed for twenty days. After this period, explants of cotyledons and hypocotyles were subjected to six different treatments containing MS-61 basic medium. The cultures were incubated at 27C (16h light / 8h dark). Media with 2-4-diclorofenoxiacetic acid (1mg/mL) and with 2-4-diclorofenoxiacetic acid (1mg/mL) plus abscisic acid (0.2 mg/mL) provided two callus lines (yellow and green, respectively) which were used for preparation of protein extracts. Callus and seeds were ground in liquid N and glass 2 powder, and the proteins precipitated with TCA/acetone solution followed by extraction and solubilization in 2-DE buffer. Protein concentrations were measured by using Plus One 2D QuantKit. The samples were submitted to 2-DE using immobilized pH gradient gels in the 3-10 pH range during isoelectric focusing. To confirm the presence of the cytolisin in callus and seeds 2-DE gels, immunoblotting was made using enterolobin antibodies raised in rabbit. Comparative analysis of 2-DE maps showed different expression between seeds and callus. SDS-PAGE followed by immunoblotting did not indicate the presence of enterolobin in callus extracts. In seeds, enterolobin five isoforms were found within the 5 to 6 pI zone.

ASSUNTO(S)

biologia molecular eletroforese bidimensional enterolobina enterolobium contortisiliquum calos vegetais proteômica

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