The Differences Of Grain Protein Content And Comparative Proteomics Analysis Between Two Genotypes Of Barley Under Different Nitrogen Levels

To investigate the molecular mechanism of the barely grain protein content with different applications, Yangsimai 3, Naso Nijo and thirty-eight barley cultivars were used in this study. Yangsimai 3 is a Chinese landrace of feed barley, two-rowed, with a high grain protein content (GPC), Naso Nijo is a Japanese two-rowed malting barley cultivar with a low GPC. Grain protein contents, albumin content, globulin content, hordein content and glutelin content were measured. Proteome profilings of differentially expressed protein were established by using a combination of 2-DE and MALDI TOF MS. The main results were summarized as follows:1. Grain protein contents were measured between mature grains of Yangsimai 3 and Naso Nijo. A proteome profiling of differentially expressed protein was established by using a combination of 2-DE and MALDI TOF MS. In total,502 reproducible protein spots in barley seed proteome were detected with a pH range of 4-7 and 6-11, among these 41 protein spots (8.17%) which were detected differentially expressed between Yangsimai 3 and Naso Nijo. The number of protein spots defined as quantitative and qualitative differences was twenty-one and twenty, respectively. Thirty-four protein spots corresponding to twenty-three different proteins were identified, which were grouped into eight categories, including stress, protein degradation and posttranslational modification, development, cell, signalling, glycolysis, starch metabolism and other functions. Among the identified proteins, enolase (spot 274) and small subunit of ADP-glucose pyrophosphorylase (spot 271) were exclusively expressed in barley Yangsimai 3, which may be involved in regulating seed protein expression. In addition, malting quality was characterized by an accumulation of serpin protein, alpha-amylase/trypsin inhibitor CMb and alpha-amylase inhibitor BDAI-1.2. Differentially expressed protein profilings with albumin, globulin, hordein and glutelin were constructed between Yangsimai 3 and Naso Nijo under different nitrogen levels, a total of 152 protein spots were found to be differentially expressed with a significant difference at p<0.05.108 protein spots were identified by using MALDI TOF MS, which were involved in thirteen functional categories, including fermentation, signalling, tricarboxylic acid cycle, redox, metabolism, degradation, RNA, photosynthesis, stress, protein degradation and posttranslational modification, glycolysis, development, and other functions. Differentially expressed proteins of albumin and globulin mainly consisted of development, metabolism, protein degradation and posttranslational modification, glycolysis, tricarboxylic acid cycle and redox, which involved in signal transduction and energy transformation. Hordein and glutelin were mainly contributed to accumulation of seed storage proteins and protein degradation and posttranslational modification. The expression of enolase, glyceraldehyde 3-phosphoric acid dehydrogenase and glutamic dehydrogenase displayed up-regulated, which were involved in photosynthesis, respiration, metabolism and transformation process. Peroxidase and glutamate dehydrogenase were the main enzyme for amino acid synthesis and transformation process in barley, involving in the ammonia assimilation and impact on plant nitrogen metabolism. With the increase of nitrogen fertilizer, the expression of these enzymes increased. While 14-3-3 proteins was down-regulated and associated with carbohydrate metabolism. The differentially expressed proteins may be contributed to the nitrogen regulation of grain protein contents in barley.3. Grain protein contents and hordein contents of 38 barley varieties were measured; SDS-PAGE and protein expression profilings were contructed. The results displayed that the grain protein contents ranged from 11.13%-18.17%, hordein contents ranged from 3.74%-8.26%, the proportion of hordein and grain protein contents ranged from 32%-50%, which indicated that hordein were the major protein fraction in grain protein. Eighteen types of hordein SDS-PAGE patterns were observed in the tested materials. To construct a B-hordein expression profilings between barley OR71 and Franklin by using 2-DE, fifteen and thirteen protein spots were identified, respectively. Among which seven protein spots were identified as the same protein. Mass spectrometry analysis showed that these spots were B-hordein and y hordein subunits. In addition, a expression profiling between barely OR71 and E173V047 with same SDS-PAGE pattern and different hordein content was analyzed, the result showed that B hordein protein concents were resulted from expression on the protein level.