| Intrinsically disordered proteins(IDPs) is also referred to as Intrinsically Unstructured Proteins(IUPs), which is a term used to describe proteins that do not have a well-defined tertiary structure. There are two categories, complete disordered proteins and partially disordered proteins based on the degree of disordered structure. IDPs play important role in the process of physiological and pathological in organism. IDPs possess both binding promiscuity(ability to interact with multiple partners) and binding plasticity(ability to undergo binding-induced folding to accommodate diverse binding sites for different partners). IDPs usually act as hub proteins which interact with multiple partners to connect various biological molecules in protein interaction networks. In plant, IDPs usually take part in signaling, transcription regulation facilitates phosphorylation, molecular chaperone and molecular shield. Based on the research of the LEA protein family, GRAS protein family, IDPs was taken part in response to external environment and stress signal in plant. Previous studies of these proteins mainly focus on the physiological function of single IDP in plants, yet no comprehensive study on the IDPs related to resistance to drought and stress has been reported so far.In our previous studies,We found that, the survival ratio of R0(the stage before radicle break the seed coat) after dehydration is 83%, while R15(the radicle outside the seed coat is 15mm) is almost zero. This means that R0 is desiccation tolerant,and R15 is desiccation sensitive. In this study, the soybean radicles at R0 and R15 stage were collectedand heat stable proteins were extracted for i TRAQ-ESI-MS/MS. The result indicated that among the 4132 proteins detected by mass spectrum, 795 differential proteins were obtained after statistical tests. among them, 170 proteins was up-regulated in R0 stage, and 89 proteins were up-regulated in R15 stage. By disordered analysis by Espritz online software, the result indicated that among the 795 differential proteins, the rate of 100% disordered proteins and 60~90% disordered proteins rate were 3.01% and 11.82% respectively, which was far more higher than the 0.96% and 7.87% in soybean total proteins. This result indicated that, after heat treatment, >60% disordered proteins were well concentrated. Among which, 60.6% of the R0 stage high expressed proteins were >60% disordered; while only 22.4% of the stable proteins and 12.4% of the R15 stage high expressed proteins were >60% disordered. The above results indicat that there were more IDPs in the tissues with high resistance to stress, the IDPs might be related to the resistance to dehydration stress at R0 stage.We utilized egg NOG database, GO database and KEGG database to conduct function annotation on differential proteins, and the result from egg NOG annotation indicated that there were more proteins participating in transcription, ribosome structure, biosynthesis, posttranslational modification, protein transportation, chaperone and carbohydrate transportation and metabolism, amino acid transportation and metabolism and other functions. The annotation resulted from GO database indicated that the proteins were enriched in 6 entries: nutrition accumulation, translation elongation, protein aggregation, water response, embryonic development and glycine metabolism. The result from the KEGG database indicated that the proteins were enriched in methionine metabolism pathway and phenylalanine metabolism pathway, indicating that the heat stable proteins may be involved the process of antioxidation during seeds germination.We selected 10 genes from Ro stage high expressed proteins and stable proteins respectively to design the primers. The secondary structure of 10 proteins was detected by circular dichroism spectroscopy, among which there were 4 proteins possessing classical randon coil structure, namely transcription activator-related(NO.1), m RNA splicing factor(NO.5), NO.9 c AMP-regulated phosphoprotein 19-related protein(Arpp19) and NO. 10 PM35. The other 6 proteins showed α-helical or β-sheet structure in PBS buffer.Through the comparison of the protective function of these 10 proteins on LDH enzyme under repetitive freeze-thawing stress conditions, we found that, compared with lysozyme, all the ten proteins could protect LDH at the molar ratio of 1:1, 5:1,10:1(protein:LDH). Among them, HSBP showed the strongest protective ability among the 10 proteins. The results show that the ability of the protein on protecting LDH enzyme activity is not positively correlated with the content of disorder. We speculated that the IDPs protein can protect target protein through themechanism of molecular shield and inhibition of target molecule collision while the folded proteins protect target protein through reducing the ice crystals. |
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