Characterization Of The Physicochemical Properties Of Maize Protein EMB564Based On Bioinformatics

To identify specific proteins related to maize seed viability, seeds of Zhengdan958were sorted based on viability evaluation with triphenyltetrazolium chloride (TTC) assay and the embryos used for comparative proteomic analysis. It is found that the abundance of EMB564protein, belonging to the LEA protein superfamily, is significantly different in embryos between high-vigor and low-vigor maize seeds. Based on bioinformatics, we analyzed the amino acid sequence of the EMB564protein, prepared peptide antibodies for the conserved domain, predicted subcellular localization and researched the physicochemical properties. In addition, we established a method of improving gel-based proteome analysis of soluble protein extracts by heat prefractionation in researching the thermal stability of EMB564protein. The main results are represented below:1Proteomic analysis of high-vigor and low-vigor maize seeds embryosComparative proteomic analysis revealed that28protein spots identified were differently expressed significantly between high-vigor and low-vigor maize seeds embryos, of which20were up-regulated and8down-regulated in R embryos. Among them were proteins involved in stress response, protein folding, and stabilization, as wells as proteins related to nutrient reservoir and metabolism. Prominently, small heat shock proteins, late embryogenesis abundant (LEA) proteins, and antioxidant enzymes were highly up-regulated, while two proteases were highly down-regulated in high-vigor embryos compared to low-vigor embryos. One of LEA proteins was EMB564, which content a large number in high-vigor seeds but none could be detected in the loss of seed vitality. Further found that the artificial aging experiments EMB564lower content consistent with the process of seed deterioration2Cellular localization of EMB564proteinUsing peptide antibodies against the EMB564protein conservative domain, we compared the content of EMB564protein between plumule, radicle and scutellum by western blot. The result showed that the EMB564protein exists in plumule, radicle and scutellum, while it’s content is difference in different tissues, plumule has highest concentrations, followed by radicle, scutellum at least.Subcellular Localization of EMB564protein by immunogold electron microscopy technique in maize plumule, defined it mainly on the surface of the chromatin in the nucleus, which is consistency with the predicted three-dimensional structure suggesting combined with DNA.3Characterization and application of the physicochemical properties of maize protein EMB564The soluble protein of maize seed embryo is divided into supernatant (heat stable protein) and precipitated (two parts of the heat-labile protein) by95℃heating. The EMB564is heat stable protein by SDS-PAGE and Western blot analysis. We found that EMB564protein could be enriched in supernatant by heat prefractionation.On the basis of protein thermostability differences, we established the method of improving gel-based proteome analysis of soluble protein extracts by heat prefractionation, which can enhance the detection of low-abundance proteins and improve the resolution of2DE. Soluble proteins were extracted from maize embryos and leaves. Through heating at95℃for5min, soluble protein extracts were prefractionated as heat stable protein fraction (the supernatant) and heat labile protein fraction (the precipitate). Our results showed that heat prefractionation enhanced the separation of proteins in both fractions by2DE, thereby increasing the chance of detecting low-abundance proteins, many of which were nonvisible in unfractionated extract. In maize embryo,330spots were detected in soluble protein extract, while577spots were detected after prefractionation. Furthermore, this prefractionation method facilitated the enrichment, detection, and identification of de novo synthesized stress proteins.EMB564protein can be enriched by heat prefractionation, and then the supernatant fraction containing EMB564protein incubated with hydroxylapatite in the centrifuge tube. Proteins can be divided into two parts:adsorpted by hydroxylapatite and don’t. SDS-PAGE analysis shows that the EMB564protein can’t be adsorpted by the HAP. Using the characteristic, we established an efficient method to purify a large number of EMB564protein. Compared the changes of CAT activity in the crude enzyme solution with or without exogenous EMB564protein after50℃heating5min. Preliminary experiments found that EMB564protein in vitro protects the activity of CAT from heat stress, and the effect is dependent on the concentration of the exogenous EMB564protein.