Evaluation of protein and metalloprotein changes in soybean seed after genetic modification process / Avaliação das alterações proteicas e metaloproteicas em soja apos o processo de modificação genetica

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

This research work consisted of comparison of two kinds of soybeans [Glycine max (L.) Merrill], transgenic and non-transgenic, using proteomics and metalloproteomics changes. Firstly, soybeans proteins were extracted with an extraction buffer containing Tris-HCI (pH 8.8), KCI, DTT, PMSF and SDS, and then they were quantified by the Bradford method. In terms of total protein concentration, both kinds of soybean seed showed a similar result: 8.2 ± 0.3 mg mL for transgenic and 11.0 ± 0.1 mg mL for non-transgenic soybeans. Afterwards, proteins were separated by 2-D PAGE. The electrophorectic process was optimized, in order to obtain gels with a good resolution, and the proteomic profiles of both kinds of soybeans evaluated were compared. In gels having the pl range 4 to 7, transgenic soybeans showed, on average, a greater number of protein spots in the gel (408 ± 27) than did the non-transgenic soybeans (397 ± 26) and the probability of identity between these protein groups was only 35.0%. In another step, 10 protein spots were cut out from the polyacrylamide gel, digested with trypsin, and subsequently characterized by MALDI-TOF MS. LEA protein and early nodulin 75 (precursor) showed differences in their expression, as observed by the different normalized volumes of their spots in the electrophorectic gels of each kind of soybeans. Metal ions such as Ca, Cu, Fe, Mn, Ni and Zn were mapped in some protein spots using the SR-XRF technique, which detected a greater number of metal ions in transgenic soybeans. Calcium, copper and iron were quantified with an atomic absorption spectrometry technique (ETAAS or FAAS) in the proteins where those metal ions were detected by qualitative analysis by SR-XRF. Prior to quantification, protein spots were decomposed by microwave assisted decomposition in mini-vials, a process that was also optimized in this work. Mostly, the results of quantitative analyses agreed with those obtained by qualitative analyses as well as with the information available in the protein data bank about the presence of metal ions in the proteins whose characterization was done. The exceptions found are related to the proteins glycinin G2 (precursor), in which the presence of iron was confirmed, and chalcone synthase 2, in which the presence of calcium, copper and iron was confirmed. Such data, however, are not available in the protein data bank.

ASSUNTO(S)

metaloproteinas soja proteinas proteins genetic modification soybean seed modificação genetica metalloproteins

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