Characterization Of Y Type HMW Glutenins In Two Species Of Leymus

High molecular weight glutenin subunits (HMW-GS) were important storage proteins in Triticeae species. The compositions and contents of HMW-GSs play an important roles in determining the end-use quality of wheat flours. The HMW-GSs and their coding sequences in Leymus species were determined by using SDS-PAGE, gene cloning, sequencing and bacterial expression. The main results were as follows:1. HMW-GS compositions in Leymus speciesThere were1-3putative HMW-GSs in each of the six accessions from four Leymus species. The electrophoretic mobility of candidate HMW-GS in Leymus species was very similar to those of subunits in P. juncea, possibly suggested that these proteins were encode by Ns genome of Leymus species. Because there was no defined Xm genome diploid donor, the electrophoretic mobility of HMW-GS encode by Xm genome could not confirmed.2. Molecular cloning and bacterial expression of HMW-GS genes from L. racemosus ssp. racemosus and L. chinensisOne and two DNA fragments were amplified from L. racemosus ssp. racemosus W623305and L. chinensis PI499516. Sequecing results verified three y type gene ORFs, Racy, Chiyl and Chiy2. The DNA lengths of them were very small, with1,272(Racy)、1,389(Chiy1) and1,234bp(Chiy2), respectively.Racy and Chiy2share similar structural domains as typcial y types. The N-termianl length of Chiyl was reduced to99aa residues by absents an18bp DNA fragment and its encoded aa residues EGEASR at the starting of N-terminal. Five other y type genes, inclduing those from Elymus ciliaris, Pseudoroegneria libanotica and Leymus mollis, were similar to Chiyl. Sequencing suggested that Chiyl and Racy had intact ORFs and could be predicte to mature HMW-GS with small molecular sizes. Bacterial expression of the modified ORFs (without signal peptides) of Chiyl and Racy confirmed expression of HMW-GSs. The ORF of Chiy2was probably not expressed because it had a two-base deletion in its N-terminal, resulting in a frame-shift mutation and indirectly producing downstream in-frame stop codons (TAG).3. Phylogenctic relationshipsA maximum likelihood tree was constructed using the aa residues in signal peptide, N and C-terminal. The y-type HMW-GSs in Triticeae formed two clades. The first contained Uy, Cy, Eel.8y, Ky, Gy, Tay, and Ry from Aegilops ssp., Elytrigia elongata, Crithopsis delileana, Taeniatherum crinitum, Secale cereale, and Triticum ssp. The y-type HMW-GSs from O, Q, W, V, P, St, F, Xe, and H from tetraploid Leymus and Elymus formed the second clade. The y-type HMW-GS lacking the EGEASR residues at the start of the N-terminal formed a subclade within the second clade.