Acúmulo de ricina em sementes de mamona e silenciamento do gene em plantas geneticamente modificadas

AUTOR(ES)
DATA DE PUBLICAÇÃO

2010

RESUMO

Castor bean (Ricinus communis L.) is an oilseed of great economic and social importance. The oil extracted from its seeds is used in biodiesel production and as raw material for chemicals, cosmetics and lubricants. Besides the oil extracted from the seeds, their production chain generates side-products, which can be used as organic fertilizer of good quality (high nitrogen concentration) or as animal food (with high protein content). The main obstacle to its use for animal feeding is ricin, a highly toxic protein, found in the seed. Ricin is a lectin composed of two subunits known as chains A and B. When ingested, the B chain binds to galactose on the cell surface, penetrating into the cell. Inside the cell, the A chain, which has enzymatic activity, inactivates the 60S subunit of the ribosomes preventing the production of proteins, leading to cell death. The objectives of this study was (1) to assess the existence of genetic variability for the concentration of ricin in castor bean seeds from 20 accessions of germplasm bank of Embrapa, (2) study the distribution and accumulation of ricin during seed development, and (3) perform genetic transformation aimed at silencing the genes coding for ricin in genetically modified castor bean seeds. ELISA (Enzyme-linked immunosorbent assay) was used to quantify ricin in total protein extracts of seeds, immunocytochemical analysis using light and electron microscopy for the localization of ricin in cells of the endosperm of the seed and genetic transformation by biolistic targeting the silencing of this protein in castor seeds, using the strategy of interfering RNA (RNAi). The results showed the existence of variability for the concentration of ricin in accessions of the germplasm bank of Embrapa Algodão, it is possible to select genotypes with high and low levels of ricin. The cultivars IAC Guarani, BRS Nordestina and BRS Paraguaçu had the lowest concentrations of ricin, and are quite suitable for breeding aiming to use the seed cake to feed animals. Among the commercial cultivars, BRS Energia presented the highest concentration of this protein. During the development of cells in the endosperm of castor seeds of BRS Energia exhibited an increasing number of reserve vesicles (protein storage vacuoles and lipid bodies) and decreased cytoplasmic volume. The accumulation of ricin in the BRS Energia increased with seed development, and in the mature seed (60DAP) the concentration was the maximum. Ricin can also be found in crystalloids of protein storage vacuoles (PSV) and not only in the matrix, contrary to previous biochemical observations that the protein components were found only compartmentalized within the protein bodies. Genetic transformation of castor bean was developed, with the regulator TDZ being more effective in inducing shoots then BAP. In addition, IBA and AgNO3 were important for the elongation of shoots and rooting of explants. The use of bioreactors in order to elongate the explants was effective for short periods. It is possible to obtain genetically modified castor bean using as selective agent the imazapyr. The transformation system based on the biolistic process presented efficiency similar to other protocols.

ASSUNTO(S)

biologia molecular-proteínas sementes-toxocologia biologia molecular mamona-genética vegetal

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