Characterization Of A Novel Y-type HMW-GS With Eight Cysteine Residues From Triticum Monococcum Ssp. Monococcum

T. monococcum ssp. monococcum (A~mA~m,2n= 2x= 14) contains many valuable genes that can widen the genetic bases of wheat and plays a key role in improving the quality of wheat. The composition and number of high-molecular-weight glutenin subunits (HMW-GSs) play important roles in determining the grain-processing quality of common wheat. The Glu-Aly allele is always silent in common wheat. However, this subunit is always active in many species of diploid wheat and tetraploid wheat. It is difficult to transfer Glu-Aly allele from T. monococcum ssp. monococcum into wheat by direct cross. Amphidiploid artificially doubled from F1 hybrids between Triticum turgidum and T. monococcum ssp. monococcum could be used as a bridge to transfer lAy8.2 into common wheat cultivars. In this study,1Ay gene of Triticum monococcum. ssp. monococcum accession PI560726 was characterized by SDS-PAGE, gene cloning and bacterial expression. The main results are as follows:1. SDS-PAGE analysis revealed that the T. monococcum ssp. monococcum accession PI560726 expressed an active y-type HMW-GS. The electrophoretic mobility of the y-type subunit was slightly faster than that of the 1By8 of the common wheat cultivar Chinese Spring and differed from those of the previously identified 1Ay genes. Based on its electrophoretic mobility, the y-type subunit was designated 1Ay8.2.2. The active y-type HMW-GS termed lAy8.2 was cloned and sequenced. The sequence was submitted to Genebank (No. KP137569). Compared with previously reported active 1Ay subunits, this novel subunit contained an extra cysteine residue at position 103 of the amino acid sequence in the N-terminal region, in addition to the six cysteines in the N-and C- terminal regions found in most active lAy subunits and the one in the repetitive region that appears in only a few 1Ay alleles.3. A bacterial expression experiment was conducted to confirm that the cloned Glu-Aly ORF sequence accurately corresponded to the subunit in the seeds. The expressed mature protein showed an eletrophoretic band identical to the 1Ay8.2 from seed of T. monococcum ssp. monococcum accession PI560726.4. To further investigate the evolution of the alleles encoded by Glu-A1-2, a neighbor-joining tree was constructed. The results revealed that the Glu-Al-2 alleles were clustered together with the exception of the 1Ay gene EU984508 from T. monococcum ssp. aegilopoides. The lAy8.2 subunit clustered together with the 1Ay genes EU984506 and JQ318695, which have seven cysteines.5. lAy8.2 subunit was expressed in an amphiploid (AABBA~mA~m,2n= 6x= 42) between T. turgidum. ssp. dicoccon and T. monococcum ssp. monococcum PI560726. This amphiploid could be used as a bridge to transfer lAy8.2 into common wheat cultivars. Replacing the silenced 1Ay in common wheat with the active 1Ay8.2 allele harboring an extra cysteine residue is expected to improve the quality by increasing the number of HMW-GSs and promoting the formation of covalent interactions through disulfide bonds with the extra cysteine residue.