Identification And Sequence Analysis Of Glutenin Genes From Some Triticeae Species

Wheat seed storage proteins were one of the determinants of flour processing qualities,which contained two important components of Glutenins and Gliadins.Glutenins were further divided into high-molecular-weight glutenin subunits(HMW-GS)and low-molecular-weight glutenin subunits(LMW-GS),and they could form glutenin macropolymer(GMP)by interaction and further affected the dough elasticity.HMW-GS played a key role in controlling flour processing quality,and D-hordein was its homologous protein in Hordeum,both of them belonged to HMW-prolamin;The LMW-GS,accounting for 60% of total glutenins,also played an important role in determining the gluten strength.Hence,characterization of glutenins from Triticeae species was important for improving wheat processing quality.In this study,LMW-GS and HMW-prolamin genes were respectively identified from Xinjiang winter wheat landraces and the species of genera Psathyrostachys and Leymus.The main results were as following:1)The genetic diversities of 75 Xinjiang winter wheat landraces were characterized by Diversity Arrays Technology(DArT).Clustering analysis was performed according to the effective DArT markers on each of the group 1 chromosomes 1A,1B and 1D respectively or as a whole.The resultant clustering diagrams were nearly the same among four different conditions.All of the materials were divided into six groups,and 15 materials were selected for further characterizing of the LMW-GS genes.Four LMW-GS gene specific primers were used for PCR amplifications.A total of 105 LMW-GS gene sequences were identified.Eighty-eight non-redundant LMW-GS sequences were obtained after excluding redundant sequences among different materials.After blasting in the GenBank database,16 novel chimeric genes were identified,and were further divided into four groups(I to IV).The genes in groups I and II were chimerism of two genes from the same and different loci,respectively.Similarly,the genes in groups III and IV were chimerism of three genes,which were separately from two and three loci.In group I,active gene DM1839-3 was the chimerism of two pseudogenes GluA3-2 and GluA3-391,suggesting that chimerism may contribute to increase the expressible LMW-GS gene numbers.Phylogenetic analysis revealed that,of the 16 chimeric genes,nine genes were clustered together with known genes from the Glu-3,while the remaining seven genes were different from known genes and formed relatively independent branch.2)Three novel D-hordeins,Ns 1.3,Ns 2.6 and Ns 2.9,were isolated from Psathyrostachys juncea PI 315080.Ns 1.3 differed from Ns 2.6/Ns 2.9 by having a shorter open reading frame(<1.5 kb versus >2.5 kb)and was probably not expressed as a normal protein,while Ns 2.6 and Ns 2.9 was verified by bacterial expression.By comparing with Triticeae HMW-GS and D-hordein of Hordeum,the Ps.juncea D-hordeins Ns 2.6/Ns 2.9 shared highly similar primary structures with them,but had a larger molecular mass,longer repetitive domain and more cysteine residues(nine in total)than Triticeae HMW-GS,and a longer N-terminal length than D-hordein of Hordeum,those characteristics may have the potential to be very important for improving the end-use quality of wheat flours,in addition to contributing to the understanding of the evolution of Triticeae HMW-prolamin.Phylogenetic analysis revealed that the Ps.juncea D-hordeins were divided into Ns 1.3 type and Ns 2.6/Ns 2.9 type.3)Nine novel D-hordeins,Lmul 1.2,2.4,2.7,1.3-1,1.3-2,1.3-3 and Lchi 2.5,1.3-1,1.3-2,were isolated from Leymus multicaulis and L.chinensis.Four genes,Lmul 1.2,2.4,2.7 and Lchi 2.5,were verified by bacterial expression,while the other five sequences were pseudogenes.Four D-hordeins from two Leymus species had a longer N-terminal length than those of the Hordeum species(116/118 vs.110 amino acid residues,AA residues),while three of them(Lmul 1.2,2.4 and 2.7)had 116 AA residues in the N-terminal,which were two AA residues less than those of Ps.juncea.Besides,an additional cysteine was identified from Lmul 1.2 and 2.7,and Lmul 1.2 was also identified as the smallest D-hordein due to the shortest coding region.Phylogenetic analysis revealed that the nine Leymus D-hordeins were evolved into two independent clades,with all the 1.3 types clustered with Ps.juncea Ns 1.3,and the others aggregated with the D-hordeins from Hordeum and Ps.juncea and the HMW-GSs from Leymus.Within the Clade of HMW-GSs and D-hordeins of various species,the Leymus HMW-GSs formed an independent branch intermediated between the D-hordeins from Ps.juncea and Leymus.These results provided potential candidate genes for the improvement of wheat processing qualities,and also provided basic information for understanding the distribution of D-hordeins in different wheat species.