Molecular Identification Of Genes Encoding AGPase,the Key Enzyme Involved In Starch Synthesis In Wheat And Its Relatives

Common wheat?Triticum aestivum L.,2n=6x=42,AABBDD?is one of the most important cereal crops in the world.In the past,most of the researches were focused on the improvement of yield and neglected the improvement of quality.As a result,the yield of wheat in our country is huge,but the high-quality wheat still needs to be imported from other countries.In recent years,more studies were focused on the wheat quality,but the yield of high-quality wheat is insufficient for the market.Starch is the most abundant storage reserve carbohydrate in wheat seed and could greatly influence the processing quality of wheat flour.For a long time,the wheat quality researches were focused on the protein and their amino acid component and neglected the effects of starch on wheat quality.The synthesis of starch relies on the cooperation among four types of enzyme.Now,sequence information and functional information of some genes including ADP-glucose pyrophosphorylase?AGP?are insufficient.AGP is the rate-limiting enzyme in the pathway of starch synthesis and could greatly influence the starch synthesis.Wheat relatives like Triticum urartu,Aegilops speltoides,Aegilops tauschii are genome donor species of wheat and play important roles for the research on wheat functional gene.Moreover,the wheat relatives possess abundant gene diversity and are important resource for wheat breeding.So,it is important to carry out the research on wheat starch biosynthesis and AGP in wheat and its relatives.The main conclusions derived from these studies include:1.In this study,amylose and amylopectin from wheat grains were isolated.The purity and applicability of amylose and amylopectin were verified by different ways.In addition,the determination procedure of wheat starch was optimized.The results showed that the composition of wheat starch could be measured in an accurate,efficient and low-cost way based on the wheat starch standards and optimized procedure.2.Based on the optimized procedure,the starch accumulation pattern in wheat grains at different developmental stages was analyzed.The results showed that the starch accumulation pattern including total starch content,the composition of starch and the accumulation rate in 4 wheat varieties were different.Generally,the total starch content and amylopectin content increased rapidly from 7 DPA?days post anthesis?to 16 DPA.The contents could be twice more than those in earlier stage and then increased slowly or trend to level off after 16 DPA.The content of amylose increased slowly during the whole developmental stage.3.At different developmental stages,the gene expression analysis showed that three types of soluble starch synthase?SSS?presented similar tendency and expression levels and all of their expression rose obviously at the middle stage?12 DPA to 21 DPA?.The starch branching enzyme?SBE?I and II increased their expression at the middle stage.The expression and changes of SBEI were higher than other two types of SBE and SBEIII showed a very low and irregular expression during the whole developmental process.Both two types of starch debranching enzyme?DBE?increased their expression rapidly at the middle stage,but the expression levels and time of duration were varied in different wheat.The expression of AGP-S gene rose rapidly at early stage and reached the peak at 12 DPA,and then maintained high expression levels with gradual decline.The expression levels of AGP-L in different Bobwhite were similar,but,the expression in Bobwhite 7 changed more smoothly than those in Bobwhite5.4.We isolated a full-length open reading frame?ORF?of AGP-L gene?named as Agp2?in wheat Bobwhite and a series of Agp2 coding region sequences in 18 wheat relatives.In wheat relatives,the coding region of Agp2 contained 15 exons and 14 introns including a full-length ORF of 1566 nucleotides and the major variation between different sequences was located in intron 2.Generally,the phylogenetic tree of Agp2 indicated that sequences from A and D genome donor species were most similar to each other and sequences from S genome species contained more variation.Starch accumulation and Agp2expression in wheat grains reached their peak at 21 and 15 days post anthesis?DPA?,respectively.Moreover,a strong correlation between starch accumulation and Agp2expression was observed.For the subsequent analysis of enzyme activity,the wheat AGP-L was successfully expressed in Escherichia coli.5.We isolated the full-length ORF sequence of the AGP-S gene?designed as Agp1?from wheat Bobwhite and gene coding region sequences from 19 relatives of wheat.In different wheat relatives,the coding region of Agp1 ranged from 5948 bp to 7666 bp and contained 9 exons and 8 introns.The full-length ORF sequence of Agp1 encoded a polypeptide containing 473 amino acids without transit peptides.The sequence alignment and phylogenetic tree analyses indicated that the Agp1 ORF sequence was highly conserved?99.7%?across the wheat relatives examined in this study.Variations in introns reduced the similar?79.5%?of Agp1 sequences from wheat relatives,and Agp1 coding region sequences from the S genome species were divergent.In developing grains,rapid increase in Agp1 expression at prophase was closely followed by the accumulation of starch,and a significant?P<0.001?correlation was observed between them.The co-expression of small and large subunits from different wheat species restarted glycogen synthesis in the E.coli AC70R1-504 cells,which are deficient in endogenous AGP activity(glg C^-/-).Our data provide useful information to evaluate and utilize Agp1 sequences in wheat and its relatives.