Otimização da extração de proteínas de raízes de feijão-caupi (Vigna unguiculata) para estudos de proteômica

Abstract

The study of proteomes in plants, is the knowledge of a set of proteins that are acting in a cell, tissue or organ, in a specific situation, such as a situation of stress, and from this can be obtained possible molecular markers. The basic steps for proteomic assays consist of the preparation of the sample, protein separation, examination and identification of proteins. However, because of the variety of plant tissues and a number of compounds that hinder the protein analysis, it is difficult to establish an efficient extraction protocol for all organs. Therefore, the purpose of this study was to optimize the protein extraction in roots of Cowpea beans already lignified based on the protocol of Mesquita (2012). Lignified and young roots were used in order to compare the efficiency of the extraction. The following treatments were performed in the lignified roots in triplicate: the use of 10 mL of extraction buffer and 1.5 g of plant material (control), 10 mL of extraction buffer and 2.0 g of plant material (10 mL x 2.0 g), 15 mL of extraction buffer and 1.5 g of plant material (15 mL x 1.5 g) and 15 mL of extraction buffer and 2.0 g of plant material (15 mL x 2.0 g); and in young plant roots: 10 mL x 1.5 g and 10 mL x 2.0 g. The roots were ground under liquid nitrogen and 10 mL of extraction buffer. The samples that were submitted to the treatment 15 mL x 1.5 g and 15 mL x 2.0 g were washed again with another 5 mL of extraction buffer. Subsequently, it was performed the solubilization, quantification, followed by an isoelectric focusing. The second dimension was carried out on polyacrylamide gel in the presence of SDS. The images of the gels stained with Coomassie G-250® (Colloidal) were scanned using the Image Scaner III (GE Healthcare®). The treatment with the highest concentration of proteins was the 15 mL x 2.0 g, followed by treatment 15 mL x 1.5 g. The control treatment and the 10 mL x 2.0 g presented the lowest results, and they did not differ between themselves by the Tukey test at 5% of probability. Regarding the sharpness and quantity of spots the results were similar. The best treatment was 15 mL x 2.0 g, followed by treatment 15 mL x 1.5 g, 10 mL x 2.0 g and 10 mL x 1.5 g. The results obtained were satisfactory, because as the volume of the extraction buffer increased, a higher efficiency of protein extraction from the sample with lignified tissues was obtained. As well as the amount of sample used was important in this optimization. Considering the obtained results, it can be concluded that the alterations in the volume of extraction buffer (2 washes) presented better results than the results that were described in the original protocol, being the treatment 15 mL x 2.0 g the one that best responded in efficiency.