Characterization Of HWM-GSs And Their Coding Genes From Triticum Monococcum And T. Dicoccoides And The Potential Utilization For Common Wheat

High molecular weight glutenin subunits (HMW-GSs) are the critical components of wheat seed storage proteins. They play a crucial role in determining dough viscoelastic properties and end-use quality of wheat flour. Variation in the composition and amount of HMW-GS would directly affect the flour processing quality. Cultivated einkorn wheat (Triticum monococcum, AA,2n=2x=14) and wild emmer wheat (T. dicoccoides, AABB, 2n=4x=28) contain the homologous genomes with common wheat. Extensive allelic variation of HMW-GS occurs in this two species. Therefore, isolation and molecular characterization of novel HMW-GS genes in T. monococcum and T. dicoccoides would not only provide potential elite gene resources for wheat quality improvement, but also offer useful information for further understanding the origin and evolution of HMW-GS genes. This work included gene cloning, heterogenous expression, sequence comparison, protein secondary structure prediction and phylogenetic analysis of novel alleles from T. monococcum and T. dicoccoides, and the introduction potential of HMW-GS from T. dicoccoides into common wheat. The main results are as follows:1. Two y-type high molecular weight glutenin subunits (HMW-GSs) lAy12* and lAy8* from the two accessions P1560720 and PI345186 of cultivated einkorn wheat (JT. monococcum ssp. monococcum, AA,2n=2x=14), were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The mobility of 1Ay12* and 1Ay8* was similar to that of 1Dy12 and 1By8 from common wheat Chinese Spring, respectively. Their ORFs respectively consisted of 1812 bp and 1935 bp, encoding 602 and 643 amino acid residues with the four typical structural domains of HMW-GS including signal peptide, conserved N-, and C-terminal and central repetitive domains. Compared with the most similar active I Ay alleles previous published, there were a total of 15 SNPs and 2 InDels in them. Their encoding functions were confirmed by successful heterogeneous expression The two novel lAy alleles were named as 1Ay12* and 1Ay8* with the accession No. JQ318694 and JQ318695 in GenBank, respectively. The two alleles were classed into the two distinct groups, Phe-type and Cys-type, which might be relevant to the differentiation of Glu-Al-2 alleles. Of which,1Ay8* belonged to Cys-type group, and its protein possessed an additional conserved cysteine residue in central repetitive region besides the six common ones in N- and C-terminal regions of Phe-type group, and was the second longest in all the known active 1Ay alleles. These results suggested that the subunit lAy8* of cultivated einkorn wheat accession PI345186 might have a potential ability to strengthen the gluten polymer interactions and be a valuable genetic resource for wheat quality improvement.2. A novel chimeric HMW-GS gene, ChAy/Bx, was isolated and characterized from T. dicoccoides (AABB,2n=4x=28) accession D129. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the electrophoretic mobility of the glutenin subunit encoded by ChAy/Bx was slightly faster than that of 1Dy12. The complete ORF of ChAy/Bx contained 1671 bp encoding a deduced polypeptide of 555 amino acid residues (or 534 amino acid residues for the mature protein), making it the smallest HMW-GS gene known from Triticum species. Sequence analysis showed that ChAy/Bx was neither a conventional x-type nor a conventional y-type subunit gene, but a novel chimeric gene. Its first 1305 bp sequence was highly homologous with the corresponding sequence of lAy type genes, while its final 366 bp sequence was highly homologous with the corresponding sequence of 1Bx type genes. The mature ChAy/Bx protein contained the forepart of 1Ay type subunit (the first 414 amino acid residues) and the back-end of 1Bx type subunit (the final 120 amino acid residues). Secondary structure prediction showed that ChAy/Bx contained some domains of 1Ay subunit and some domains of 1Bx subunit. The special structures of this HMW glutenin chimera ChAy/Bx subunit might have unique effect on the end-use quality of wheat flour. Here, it was supposed that homoeologous recombination might be a novel pathway for allelic variation or molecular evolution of HMW-GSs.3. Three novel HMW-GS genes, lAx,1Bx13.1 and 1By16.1, were isolated and characterized from T. dicoccoides accession D141. Of which, lAx was a pseudogene because a premature stop codon TGA occurs in position 835-837 in its coding region. In our previous research, an active 1Ay gene have been isolated and characterized from T. dicoccoides accession D141. It indicated that the Glu-Al locus of T. dicoccoides accession D141 only expresses a subunit lAy. This kind of Glu-Al allelic variation was firstly characterized in the molecular level. The Glu-Al in D141 might be used to solely investigate the function of the 1Ay subunit without the impact of 1Ax subunit. Thus, it should be an ideal genetic material for probing the quality effect of 1 Ay subunit. The ORF of 1Bx13.1 consisted of 2370 bp encoding a protein of 789 amino acid residues (a mature protein of 768 amino acid residues), whereas that of 1 By 16.1 consisted of 2154 bp encoding a protein of 717 residues (a mature protein of 696 amino acid residues). SDS-PAGE analysis showed that the subunit pair 1Bx13.1+1By16.1 from T. dicoccoides accession D141 had a similar migration as the superior subunit pair 1Bx13+1By16 from common wheat cv. Jimai20. Protein secondary structure prediction showed that the N- and C-terminal domains of 1Bx13.1 possessed more a-helixes than those of 1Bx13, and the a-helixes of 1By16.1 contained more amino acid residues than those of lByl6, respectively. So, the subunit pair 1Bx13.1+1By16.1 from T. dicoccoides accession D141 might have a better bread-making quality effect than the subunit pair 1Bx13+1By16 which have been proved with superior bread-making quality. In addition, we also detected the lAy and 1Bx13.1+1By16.1 from some F1 and F2 seeds of three crosses of T. aestivum×T. dicoccoides accession D141. This result indicated that the interspecific hybridization should be an efficient pathway for introducing the novel HMW-GS allelic variation into common wheat for quality improvement.