The Studies On Rice (Oryza Sativa L.) Soluble Glycoproteins: Purification And Identification

Glycoproteins are biological macromolecules widely exiting in the organisms, which including some of enzymes, hormones, antibodies. Phytohemagglutinine(PHA) and cell well structural proteins. Many studies have found out that plant glycoproteins have some relationships with stress-tolerance.This study chose rice as the research material, because of these reasons: First, the living cycle of rice is short, which make the collection easily: Second, as we all known, the complete genome sequence of rice have been finished nearly, only a little late than Arabidopsis thaliana, so its meaningful to purify the rice glycoprotein for the further studies on Proteomics Research of rice glycoproteins. In fact, we have done some preparation on this issue.The total proteins was extracted from rice leaves, and quantified by Coomassie Brilliant Blue G-250. 2-D tech was also used to display the pattern of total proteins, which is the base of next-step separation and purification. We got 4 classifications from rice total proteins by using Ammonium Sulfate Precipitation, which was very important because we got easy way to separate and classify the total proteins. Also, it was useful for the purification of glycoproteins. The 4 classifications then purified by using ConA-Sepharose 4B Affinity Chromatography. the liquid of glycoproteins was collected by tubes in the same time of the appearances of absorption peaks. We even got some dry samples by using vacuum freezing and drying technology. Next, we done SDS-PAGE and determination respectively by Periodic Acid Schiff(PAS) staining and Coomassie Brilliant Blue(CBB) R-250 staining. In the gel of CBB staining, there are near 30 strips of different glycoproteins(some are glycopeptides) in the last 3 classifications; but the gel of PAS staining only showed 7 strips of different glycoproteins(some are glycopeptides) only because of the sensitivities of PAS much lower than CBB staining. The last step, we choose 3 glycoproteins showed on the gel of 50-80% glycoproteins classification, all of them have higher contents than others. We further purified each of the 3 glycoproteins directly from the SDS-PAGE gels. They are proved purified glycoproteins or glycopeptides by CBB and PAS staining again. The three glycoproteins are named respectively as RG1. RG2 and RG3.