| Study on the seed storability of the indica hybrid rice combinations which large-scale cultivated in Gongdong was carried out in Golden Rice seeds Co., Ltd. From the tested combinations, two combinations were screening to the further experiments, Boyou998as the storable combination and Ⅱ you998as the intolerance storage one. The physiological indexes of the two combinations were determined before storage and2years after storage, including soluble sugars and malondialdehyde (MDA) content, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity. And then, embryo protein of the two combinations was extracted for proteomics analysis by two-dimensional electrophoresis (2-DE) and MOLDI-TOF/TOF mass spectrometry. Quantitative proteomics was also conducted by iTRAQ techniques. The results showed that:1. After two years storage, germination rate between Boyou998and Ⅱ you998was very significant difference, Boyou998was80.25%and Ⅱ you998only50.25%. The soluble sugar content of Boyou998was very significantly lower than that of Ⅱ you998, not only before storage but also2years after that. And more, it did not change significantly for Boyou998, but decreased significantly for Ⅱ you998during storage. SOD and CAT activity of Boyou998was similar to Ⅱ you998, but the POD activity of Boyou998was lower than Ⅱ you998, indicating that the seed physiological metabolism of Boyou998was weaker than Ⅱ you998, might be correlated with seed vigor maintaining. The MDA content of the two combinations was not significantly different before storage, but it was very significantly higher in Ⅱ you998than in Boyou998after2years storage. Less MDA product implied that lower degree membrane lipid oxidation, and fewer harmful substances accumulated during storage, maybe also related to seed storability.2. Storable relative proteins were obtained by2D-PAGE combined with mass spectrometry and bioinformatics analysis. About2000spots were obtained on each gel pattern through two-dimensional electrophoresis gel scaning. And2-DE images were analysed by PDQuest8.0software, the number of3times (T-test95%) difference spots was94. Among these spots,74spots were analysed and identified by MOLDI-TOF/TOF mass spectrometry, except for23duplicate spots,51unique proteins were identified. For Boyou998, few proteins (6) expressed differentially before storage and2years later, and2proteins were identifed, the percentage of the total number (74) was2.70%. But for Ⅱ you 998,40proteins expressed differentially,32proteins were identified, the percentage of the total number (74) was43.24%. It also showed that the protein change of the storage sensitive combination was large, exuberant life activities resulting in large storage substance consumption.3. Before storage,28proteins were identified between Boyou998and Ⅱyou998, including19proteins raised and9proteins reduced for Boyou998. And28different proteins were identified between Boyou998and II you998after2years storage,9proteins upregulated and3proteins downregulated for Boyou998. GO enrichment analysis was conducted for the different proteins to describe the properties of the relevant genes and gene products, including molecular function, cellular component, biological process. Major proteins related to rice seed storability were determined, including LEA proteins, protease inhibitor, DNA-damage-repair/toleration protein-like, glutaredoxin, germin-like protein, cupin family protein and phosphoglycerate kinase, etc.4. The proteins related to seed storability were obtained by ITRAQ quantitive proteomics.1557proteins were identified by iTRAQ technology. For Boyou998, the number of different proteins more than3times was only5(1upregulated,4down) between before storage and2years later, but for II you998, it was20(2downregulated,18upregulated). The different proteins in Ⅱ you998were more than those in Boyou998, the result was similar to2-D proteomics.30different proteins which more than3times (26upregulated,4down) between the two combinations were determinded before storage, it was22(21upregulated,1down) between them2years after storage. And the different proteins identified by the two technique of ITRAQ and2-D were not same.Compared to all the identified proteins in the background, the major biological functions of the different proteins were determined through GO function significant enrichment analysis. At the same time, the most important biochemical metabolic pathways and signal transduction pathways were determined by the KEGG pathway application of the hypergeometric test, for the proteins related to storability.5. The preliminary mechanism of rice seed storability was put forwarded by2-DE and iTRAQ proteomics analysis. The LEA proteins, protease inhibitor and DNA-damage-repair/toleration protein-like in Boyou998were more than in II you998. These proteins are important to improve seed anti-deterioration capacity, reduce DNA damage and the consumption of storage substance, and more glutaredoxin and germin-like protein product for Boyou998to reduce the harm of reactive oxygen species(ROS). Higher cupin family protein and lectin in Ⅱ you998induce programmed cell death (apoptosis). DNA-damage-repair/toleration protein-like of Ⅱ you998decreased significantly during storage, less resilience proteins, such as SGT1proteins, base repair proteins and so on, leading to the seriously seed deteriorate of Ⅱ you998. More different proteins conducted in storage process, and the enzymes related to sugar metabolism, purine metabolism and fatty acid metabolism was higher, resulting in vigorous physical activity and more material consumption, also related to intolerance storage of Ⅱ you998seeds.6. The proteins number identified1557by iTRAQ indicated its higher accuracy, eight samples can be compared in a single experiment by iTRAQ technique showed its higher efficiency. But2-D proteomics can not be ignored, it has intuitive, reliable advantages to analyse the proteins with big difference (more than100times qualitative difference). So that the two different proteomics research method are complementary, can not be suppressed. |
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