Characterization And Evolutional Analysis Of Seed Storage Protein Genes In Wheat And Its Related Species

Wheat endosperm is main source of protein for human,and is critical for processing quality.In wheat and its relatives,seed storage proteins are mainly composed of glutenins and gliadins.High-molecular-weight glutenin subunits(HMW-GSs) have been considered as most important seed storage proteins for wheat flour quality,and play a key role in determining wheat gluten and dough elasticity which promote the formation of the larger glutenin polymer.Compared to the limited allelic variation in common wheat,there are a lot of novel alleles in related species of wheat.Therefore,many researches have been conducted on seed protein genes in wheat related species.Using SDS-PAGE,we found that one Xinjiang rice wheat accession,Daomai2,has novel subunits 1Dx2.1+1Dy10.1, which are very rare in cultivated wheat and have never been reported.In this study,we isolate the complete open reading frames(ORFs) encoding HMW glutenin subunit 1Dx2.1+1Dy10.1 from Xinjiang rice wheat accession Daomai2.Bacterial expression has also been carried out to check the gene's function.The HMW-GS 1Ay subunits are special because they are always silent in hexaploid wheat.Relative fewer researches have been conducted on this allele when compared to other loci of Glu-B1 and Glu-D1.These informations are not sufficient to understand the expression and heredity of 1Ay subunits. We identified 1Ay subunits with different electrophoretic mobility from 141 accessions of diploid and tetraploid wheats,and obtained the complete ORFs and 5' flanking sequences of 1Ay genes including.Furthermore,the 5' flanking sequences were characterized from wild diploid species of Triticeae.In addition,phylogenetic relationship of 25 Triticeae species was also analyzed by characterized promoter sequences.Due to excellent agronomic traits and high resistance to diseases,1BL/1RS translocation is extensively applied in wheat breeding programs.However,1BL/1RS translocation possesses a poor baking quality.And some defects in quality parameters such as significant reduction of dough elasticity, tenacity and strength have been accompanied with the advantages of R chromosomes.Theω-secalins is regarded as the cause of poor quality in 1BL/1RS translocation.Our knowledge about gene structure and evolution of this gene family is limited and insufficient.Therefore,we focused on characterizingω-secalins from different species involving R chromosome.1.HMW glutenin subunits from Xinjiang rice wheat accession Daomai2 were separated by SDS-PAGE.Daomai2 has four expressed HMW glutenin subunits.Subunit 1Dx2.1 migrated faster than 1Ax1,whereas slower than 1Dx2.Subunit 1Dy10.1 migrated slower than1Dy12,while slightly faster than 1Dy10.This glutenin subunit combination was very rare in hexaploid wheat.In genome PCR experiments using primers P1 and P2, four DNA fragments ranging from approximately 1.8 to 2.5 kb were amplified for Xinjiang rice wheat accession Daomai2.And four fragments(whose sizes were about 1.8, 1.97,2.2 and 2.5 kb,respectively) were cloned into the plasmid vector pMD18-T.By restriction enzyme digestion analysis and two terminal sequencing,the 2.5 kb and 1.97kb fragment were decided to represent ORFs of 1Dx2.1 and 1Dy10.1,respectively.The HMW glutenin subunit gene 1Dx2.1 has 2508 bp,encoding 836 amino acids;1Dy10.1 has 1971bp,encoding 655 amino acids.The primary structure of 1Dx2.1 and 1Dy10.1 are conserved.And mature subunits consist of three distinct domains,a non-repetitive N-terminal domain,a non-repetitive C-terminal domain with 42 residues and a large central repetitive domain.The number and distribution of cysteine are also conserved in 1Dx2.1 and 1Dy10.1.The phylogenetic tree based on N- and C-terminal amino acid sequence alignment indicated that 1Dx2.1 and 1Dy10.1 were clustered with x- and y- type subunits encoded by D genome,respectively.1Dx2.1 was most related to 1Dx2.1~t from Aegilops tauschii.2.We identified 1Ay subunits with different electrophoretic mobility from 141 accessions of diploid and tetraploid wheats,and obtained the complete ORFs and 5' flanking sequences of 1Ay genes including 6 active and 3 inactive ones.The SDS-PAGE profiles of HMW-GSs showed that 1Ay subunits were differentially expressed in T.urartu,T. monococcum aegilopoides and T.turgidum dicoccon,whereas 1Ax subunits were expressed in all accessions of these 3 species.In T.urartu and T.turgidum dicoccon,1Ay subunits displayed an electrophoretic mobility similar to that of 1Dy12 subunit,1Ay subunits from T. monococcum aegilopoides migrated slower than those of T.urartu,showing a similar electrophoretic mobility with 1By8.Interestingly,1Ay subunit in one accession(PI306526) of T.monococcum aegilopoides migrated slower than all y-type subunits and 1Bx7.We also found that for the expression frequency of 1Ay subunits,diploid wheats are higher than tetraploid wheats.According to these results,9 accessions were selected for further cloning.The amplified fragments in T.urartu and T.monococcum aegilopoides ranged from1800 to 2202 bp.It is close to the size of those typical y-type HMW-GS genes except for the fragment of 2202bp.All 6 active 1Ay possess a typical HMW-GS primary structure and some novel characteristics.Each of these deduced subunits consists of a signal peptide with 21 amino acids,a conserved N-terminal region,a central repetitive domain and a C-terminal region.The conserved cysteine residue within the repetitive domain of y-type subunits was replaced by phenylalanine residue in subunits of 1Ay(Tu-e1),1Ay(Tu-e2),1Ay(Ta-e2) and 1Ay(Td-e).Particularly,1ay(Ta-e3) has an unusual large molecular weight of 2202 bp and was one of the known largest y-type HMW-GSs.The translations of 1Ay(Tu-s),1Ay(Ta-s) and 1Ay(Td-s) were disrupted by premature stop codons in their coding regions.The possession of larger molecular mass and fewer conserved cysteine residues are unique characteristics of 1Ay subunits tested in this study.The 5' flanking sequences of active and inactive 1Ay genes show high similarity and only differ in a few base substitutions and insertions or deletions.Furthermore,the 5' flanking sequences were characterized from 23 wild diploid species of Triticeae.Multiple sequence alignment showed the 5' flanking of 23 Triticeae species regions were conserved but have more variations than those of Glu-A1-2 alleles.In addition,the 85 bp deletions have been found in promoter regions of all 1Ay genes and the corresponding positions of 6 species from Aegilops and Hordeura.The defects in the coding regions(premature stop codons and insertion of large transposon-like elements) would be possibly responsible for the silencing of the 1Ay genes in diploid, tetraploid and hexaploid wheats.However,the mechanisms of gene expression and silencing are complicated and could involve the interactions of a number of factors, including specific nucleotide sequencing,chromosome rearrangement,and methylation, etc.Some mutational events in more distant distal promoter regions are possible causes for the inactivation of 1Ay genes;and more distal sequences are necessary to be examined.3.Sequence polymorphisms and phylogenetic relationships from different genomes of 25 diploid species in Tfiticeae(Poaceae) were evaluated by using the sequences of y-type high-molecular-weight glutenin promoter(y-HGP).The length of entire y-HGP sequences varied from 845 to 915 bp in the 25 species of Triticeae.Multiple sequence alignment showed that both conservations and variations were observed in the y-HGP sequences.Higher sequence conservation was detected in the regulatory elements of y-HGP.A 85 bp deletion was found in 8 species of Triticum,Aegilops and Hordeum. Several species-specific indels were identified in the y-HGPs from Psathyrostachys, Hordeum and Pseudoroegneria.The group including T.urartu,T.boeotum,Ae. umbellulata,Ae.uniaristata,Ae.speltoides and Ae.tauschii,was identified both in maximum parsimony(MP) and Bayesian trees.The species in this group had a close relationship,and represented the genomes of the Aegilops-Triticum complex.The close relationship between Pseudoroegneria and the clade of Australopyrum,Dasypyrum and Agropyron was strongly supported in the topologies of MP and Bayesian trees.As y-HGP has sufficient amount of genetic variation and is single copy region,it is useful in phylogenetic analyses of Triticeae.4.In this study,ω-secalins from rye,hexaploid triticale,and octaploid tfiticale and 1BL/1RS translocation line were characterized.SDS-PAGE analysis indicated that molecular weight ofω-secalins is around 50 kDa.And 62 ORFs ofω-secalins were characterized.The 62 sequences share identity of > 90%with each other.Multiple sequence alignments indicated thatω-secalin genes from triticales and 1BL/1RS translocation are high homologous to those originated from rye.In 62 sequences,19 of them are fuU-ORFω-secalin genes;and the rest of 43 sequences are pseudogenes.Protein sequence analysis showed that the primary structure ofω-secalins consist of four distinct domains,a signal peptide a non-repetitive N-terminal domain,a large central repetitive domain and a non-repetitive C-terminal domain.The signal peptide is composed of 19 amino acids and highly conserved except 1 base substitution in FJ561479.The N-terminal and C-terminal are also conserved.The N-terminal ofω-sccalins all start with RQL different from 4 types ofω-gliadin,KEL/ARQ/ARE/SRL.The difference among theseω-sccalins in varied accessions mainly due to the single amino acid substitution,insertion or deletion and the variation of repeat motif number.The number of synonymous(Ks) and nonsynonymous substitutions(Ka) were calculated by DNA sequence.The results of the synonymous and nonsynonymous substitutions in the obtained gent sequences indicated that the pseudogcnes contained more nonsynonymous substitutions than those full ORF genes.This is consistent with reduced selection pressure on the pseudogenes,which wcrc not active as storage proteins.