Comparative analysis of two-dimensional electrophoresis of rice endosperm and embryo proteins by using Image Master software

Rice (Oryza sativa L.) feeds more than half of the world's population. Rice is considered a model plant in biological and agriculture field research due to its small genome compared with other plants. Storage proteins in the aleurone layer of rice seeds include albumin and globulin, as well as the endosperm, prolamin, and glutelin proteins. To determine the number of proteins and identify the protein spots between the tissues of two embryo samples and rice endosperm, the protein spots were matched according to their expression level in the images obtained from two-dimensional gel electrophoresis.
The profile of proteins in the endosperm and embryo of rice seeds was determined using isoelectric focusing (IEF) and polyacrylamide gel electrophoresis(SDS-PAGE) methods. The Image Master software was utilized to analyze the two-dimensional gels. Considering the importance of local rice consumption in Iran and the significant production of this economically important crop in the country, this study aimed to determine the diversity and differences in the expression of protein spots within the linear pH range of 4 to 7. The two-dimensional electrophoresis method was employed on the Hasan-Sarai variety of rice indigenous to the region of Guilan. For this research, the cultivar being examined was procured from the Guilan Rice Research Center. Subsequent stages of the experiment, including sample separation and total protein extraction, were conducted at the Payame Noor University laboratory in Asadabad. Ultimately, the protein samples obtained were sent to the Health Research Institute at the Faculty of Pharmacy, where two-dimensional electrophoresis was carried out using an isoelectric focusing device. Finally, the extracted protein samples were transferred to the Health Research Institute of the Faculty of Pharmacy to perform two-dimensional electrophoresis using an isoelectric focusing device.
Based on the results of the two-dimensional gel analysis, a large part of the protein spots are present in the pH range of 4 to 7. The analysis of protein profile based on standard proteins with specific molecular weight and isoelectric point indicates that embryo tissue and endosperm based on isoelectric point and molecular weight showed that 284 protein spots in the embryo and 123 protein spots in endosperm have been identified. Among the identified spots, 57 common protein spots showed increased and decreased expression between the two samples. The presence of remaining protein spots in the two samples showed a significant difference between protein content in the embryo and endosperm.
Image master software was used to determine the isoelectric point and molecular weight of protein spots and expression changes between common spots. Analysis was done in protein databases and possible proteins were identified in embryo tissue and endosperm. The proteins identified in rice endosperm and embryo tissue in two-dimensional electrophoresis included grain storage proteins, glutelin precursors, acidic and alkaline glutelins, globulins, prolamins, HSPs, and hypothetical proteins. Therefore, in this way, it is possible to identify the level of expression of proteins and also their biosynthetic pathways.