Differentially Proteomics Analysis Of Grains In Elite Wheat Shaan 253 During Filling Stage

Wheat,an important food source,is the staple food for 35%-40% of the world’s people.With the improvement of the living standard,people pay more attention to wheat quality.Recently,proteomic technology has developed rapidly,which provides probability for the exploration of protein accumulation dynamics during grain development.In this experiment,the 10 quality traits of 52 starch specific varieties were tested by correlation and principle component analysis.Shaan 253,the best one among these varieties,was selected to research the dynamic changes of high molecular weight glutenin and gliadin during grain filling stage.The scanning electron microscopy was used to observe the dynamic changes of grain starch granules.Meanwhile,sobaric tag for relative and absolute quantitation(iTRAQ)was used to explore the proteomic changes during grain development(15,20,25,and 30 days post anthesis).Quantitative real-time PCR was then performed to investigate the dynamic transcriptional expression patterns of 10 representative differentially expressed genes.Subsequently,the low molecular weight glutenin subunit(LMW-GS,Q8W3V4)encoding gene was cloned.After isolated,induced and purified,the fusion protein was then integrated into the control flour to determine the quality effect.The main results are listed as follows:1.52 accessions of wheat were tested by correlation and principle component analysis.There were abundant variation types in the test materials.There were complex relationships among the 10 quality indexes of these materials,and correlations were significant or extremely significant in most observed traits.According to principle component analysis,the first four main component made the contribution of 47.99%、18.32%、11.49%、8.06% to variation,respectively.Among them,the quality of Shaan 253 was the best.Its values of ractility,max resistance,stability time,extension area were significantly higher than those of other varieties.2.Five(1,14+15,5+10)and four(7+8,2+12)high-molecular weight glutenin subunits in Shaan 253 and Chinese spring cultivars had been detected,respectively.The types of gliadin were also varies in different cultivars.All types of high-molecular weight glutelinsubunits were found in the Shaan 253 cultivar at 15 days post anthesis.Developed with the wheat grain,these bands become more and more clear.It showed that high molecular weight glutenin was accumulated with the development of wheat grain.The main bands of ω-gliadin and γ-gliadin appeared at 15 days after anthesis,but these bands were vague.Only parts ofα-gliadin and β-gliadin were observed at that time.The protein bands increased obviously and the color of them increased significantly at 20 days post anthesis,then the bands become more and more clear.It showed that gliadin accumulated continuously during the grain filling stage.The size of A-type granules increased rapidly from 15 to 20 days post-anthesis,but B-type granules grew slowly from 15 to 30 days post-anthesis.Besides,starch granules accumulated continuously until grain maturity.3.A total of 30360 unique peptides mapped to 7069 proteins which correspond to 1%false discovery rate were obtained.Among them,859 differentially expressed proteins(DEPs)showed at least 2-fold differences,including 498 DEPs detected at one stage,227 DEPs shared by two stages and 134 DEPs shared by all development stages.The 134 DEPs were divided into six function groups.Most of proteins involved in IV-VI groups showed a significant accumulation after 15 days post anthesis.4.The differentially expressed proteins identified at first,second,third periods were classified into 27,33,33 GO terms,respectively.Based on GO(gene ontology)analysis,the functions of the proteins were classified into 3 groups,including cellular components,molecular functions,and biological processes.Among these GO terms,most proteins were involved in cell,cell part,catalytic activity,binding,metabolic process,cellular process.The detected proteins were classified into 17,21,21 clusters of COG categories corresponding to first,second,third periods,respectively.The three clusters of G(Carbohydrate transport and metabolism),O(post-translational modification,protein turnover,chaperones),and R(General function prediction only)were the most important categories at all development stages.KEGG pathway analysis revealed that 24 pathways significantly enriched with association signals at the p < 0.05 level and the number of pathways at three development stages were 14,8,2,respectively.5.A large number of proteins related to energy metabolism,starch synthesis,storage proteins,stress and defense had been detected.Glyceraldehyde-3-phosphate dehydrogenase(W5ATV6)and ATP synthases(W5AMD4,W5GYE4,S4Z0A5)were closely related to energy metabolism.These proteins were gradually down-regulated at 15-30 days post anthesis.Granule-bound starch synthase(W5DNU0)and starch branching enzyme(O04074)were up-regulated and had the highest expression level at 30 days post anthesis.Storage proteins such as high-molecular weight glutenin subunit(W5AIU1),low-molecular weight gluteninsubunit(Q8W3V4),gliadin(D2KFG9)and avenin-like protein(W5DVL2)also accumulated during this period.At the same time,32 DEPs were related to stress and defense.6.Low-molecular weight glutenin subunit(Q8W3V4),a typical protein in proteomic data,was selected to clone its gene.The open reading frame(ORF)of clone sequence was894 bp and coded for a polypeptide of 298 amino acids.The sequences comparison suggested that only 2 bases were different between the open reading frame sequences of the fragment and the reported Q8W3V4 gene.The deduced amino acid sequences also had 2 sites difference with Q8W3V4.These results were further illustrated the accuracy of proteomic data.Among the three types of secondary structures,content of coil was the highest with55.37%,α-helix was the second with 35.57%,and extended strand was the lowest with 9.06%.Phylogenetic and evolutionary analyses showed that the deduced protein was closely related to LMW-GS in Triticum timopheevii,while it was far with Elytrigiarepens(Linn.)Nevski and Secalecereale.SDS-PAGE results revealed that the gene was expressed in E.coli system.The farinograph data showed that target protein has a significant positive effect on wheat dough quality.