Molecular Characterization And Functional Properties Of Prolamin Genes From Common Wheat And Its Derivate/Related Grasses

In order to exploit y-gliadin composition and possible relationship with wheat breeding, A PCR-based strategy was applied to obtain the DNA sequence of y-gliadin open reading frames present in lineâ…¡-12, a derivative from a somatic hybrid between bread wheat (Triticum aestivum L.) cv. Jinan 177 and tall wheatgrass (Agropyron elongatum,10x). A total fifty analysable sequences were obtained, eighteen fromâ…¡-12 and sixteen each from the parents. Amplicon length ranged from 720 to 936 bp, corresponding to a putative mature protein of 239-309 residues. The primary structure of these putative proteins comprised five domains. Phylogenetic analyses showed that the mature y-gliadin sequences fell into four major clades. Group 1 contained sequences shared betweenâ…¡-12 and Agropyron elongatum suggesting that some Agropyron elongatum y-gliadin genes are present in the introgression line. Group 3 has five Jinan177 and fiveâ…¡-12 sequences, indicating thatâ…¡-12 also carries wheat versions of Gli-1. Fewer genes encoding coeliac disease epitopes were present inâ…¡-12 than in the wheat donor parent.Thirteen LMW-GS genes were also characterized from a somatic hybrid introgression lineâ…¡-12. Four clones were pseudogenes because they contained an internal stop codon. The remaining nine variants contained intact open reading frames (ORFs). Sequence alignment indicates that the proteins deduced from the nine ORFs have similar primary structure with LMW-GS cloned from its parents previously. However, they have some unique modifications in the structures. The results of phylogeny showed that most LMW-GS variances fromâ…¡-12 were homologous to those from parent Jinan 177 and other wheat lines. Two LMW-GS peptides, one with a free-cysteine residue and the other with long repetitive region, were selected and heterologously expressed in E. coli and purified in sufficient scale to perform a flour supplementation test. The result showed that both of them had a negative effect to flour quality.Wild tall wheatgrass (Lophopyrum elongatum L.,2x=14) is an important resource for improving bread wheat(Titicum aestivum L.), including HMW-GS and LMW-GS relevant to end-use quality of the wheat flour. A set of fourteen distinct sequences were amplified from the genomic DNA of the tall wheatgrass, using degenerate primers targeted at Glu-3. Eleven of fourteen sequences consisted of a single intron-less intact open-reading frame. An alignment of deduced protein sequences showed that the primary structure of all eleven sequences was similar to that of wheat and other wheat-related grass Glu-3 genes. All eleven sequences carried the fourteen amino acid residue N-terminal motif MESNIIISFLK/RPWL, and were classified as LMW-m genes, based on the identity of the first amino acid of the mature protein. Phylogenetic analysis showed that the tall wheatgrass sequences were closely related to those of the intermediate wheatgrass, but only distantly so to those from decaploid tall wheatgrass. One of the eleven LMW-GS peptides with a free-cysteine residue was heterologously expressed in E. coli and purified in sufficient scale to perform a flour supplementation test. This showed that the dough strength of bread wheat flour was significantly increased by the presence of the tall wheatgrass LMW-GS.