| Rice (Oryza sativa L.), is one of the most important staple food crops for more than 60% of the human population in the world. The grain- filling characteristics of rice plays a crucial role in the yield and quality, so it is the key to study physiological and biochemical characteristics and genetic mechanism during grain filling for high yielding in rice.A large-spike rice cultivar Jinhui (indica) was used in this study. The experiment was set in 3 different proportions of nitrogen fertilizer (ratio of tiller fertilizer to panicle-grain fertilizer.7:3,6:4,5:5).The investigation was conducted to address the influence of different proportions of nitrogen fertilizer on superior and inferior grain filling of large-panicle type rice cultivars by the method of differential proteomics. The aim is to reveal the metabolic mechanism of source-sink-stream in large panicles rice during grain filling period and provide important theoretical references to dig the rice high yield potential, and to accelerate grain production increase.The results showed that there exists obvious asynchronous filling phenomenon in large -panicle type rice, and the change in the N application proportion mainly influenced the inferior grain filling, in turn altered seed setting rate and 1000-grain weight of yield components, indicating the highest grain yield at 6:4 ratio of N supply and the lowest grain yield at 7:3 ratio. Differential proteomic analysis showed much more differentially expressed proteins (26 proteins vs 9 ones in total) detected in the inferior grain than in the superior grains during grain-filling of rice under different N application proportions and 17 differential protein spots from the inferior grains and 4 ones from superior grains have been identified and assigned in functions by using MALDI-TOF/MS analysis and database searching. The result suggested that increasing N application proportion in post growth stage of rice favored in the upregulation of the genes involved in physiological activities of inferior grain filling, including the proteins associated with transport capacity of sucrose, starch biosynthesis , energy accumulation for the material metabolism, and hormone production etc in inferior grains of rice. It was also found that the increases of N application proportion in post growth stage were good for the biosynthesis of proteins and the improvement of stress-resistant ability in superior grains. The 7 key proteins to grain-filling, differentially expressed in the inferior grains mediated by different N application proportions were also detected, but no differences in expression abundance of them were found in the superior grains, which explained the molecular mechanism of environmental stability for the superior grain-filling. SPAD (Soil and Plant Analyzer Development) values of leaf of large-panicle type rice at 3 different proportions of nitrogen fertilizer applied in pre and post growth stages were investigated. The results showed that the increases of N application proportion in post growth stage were good for delaying the degradation of chlorophyll content. The method of differential proteomics were used to identify the function of differential expression proteins in leaves under 2 different proportions of nitrogen fertilizer(ratio 7:3,6:4),24 proteins were detected, and 12 proteins were identified to be the functional proteins. These proteins were classified into 4 categories according to their function. The fist group, which included chlorophyll a-b binding protein, PHY3 protein, RuBisCO activase small isoform precursor, rubisco large subunit, participated in the photosynthetic reaction. The second group was involved in the defense reactions, such as GSH-dependent dehydroascorbate reductase 1, superoxide dismutase [Mn], WRKY DNA binding domain containing protein, flavanone-3-hydroxylase.The third group was involved in the metabolism of material and energy, which include nicotinate-nucleotide adenylyltransferase family protein, aminotransferase y4uB. The fourth group participated in the material transport and signal transduction. This group included Calmodulin and Vesicle transport v-SNARE family protein. The result suggested that the effect of the increases of N application proportion in post growth stage on the leaves during the grain filling period were as follows: elongate photosynthetic time,delay senescence of leaves, strengthen the energy metabolism of leaves in post growth stage of rice, accelerate matter transportation and signal transduction.Twenty proteins with obviously different abundances were detected in leaf sheath during grain filling period under 2 different proportions of nitrogen fertilizer(ratio 7:3,6:4), and nine of them were identified in function. These proteins were classified into 5 categories according to their function. The first group involved in the photosynthetic reaction, which include ribulose bisphosphate carboxylase large subunit. The second group, which include DREPP plasma membrane polypeptide family protein, putative ATP citrate lyase a-subunit, putative endoplasmic reticulum oxidoreductin , participated in the transportation and synthesis of material. The third group involved in the signal transduction, which include Phospholipase D. The fourth group participated in the defense reactions. This group included Peroxidase , ABC-type phosphate transport system , Putative cinnamyl-alcohol dehydrogenase, Cytochrome P450 family protein. The fifth group is the Putative ATP citrate lyase a-subunit, which participated in the energy metabolism.These results suggested that increasing N application proportion in post growth stage of rice favored in physiological activities of leaf sheaf, including the photosynthesis, the transportation of material and signal, energy metabolism and resistance.The present work suggested that there existed a regulative network of complex proteome during grain filling periods and it paved the way for us to further study the molecular genetic mechanism of rice.
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