Novel High-molecular-weight Glutenin Sequences Isolated From Psathyrostachys Juncea

High-molecular-weight glutenin subunits (HMW-GSs) is an important storage protein in wheat endosperms. The composition and content of HMW-GSs play an important roles in determining the end use quality of wheat flour. Psathyrostachys juncea (2n=2x=14, NsNs) is a diploid species of Psathyrostachys. It is a perennial herb plant and exhibits cross-pollination habit. The species in Psathyrostachys contain valuable traits including cold tolerance, drought resistance and salt tolerance. Previous study of Ps. juncea has mainly focus on cytological studies including chromosome karyotype and C-banding analysis. Limited study was taken on the high-molecular-weight glutenin subunits (HMW-GSs) and their coding genes. In this study, the HMW-GSs and their coding genes in Ps.juncea were determined by using SDS-PAGE, gene cloning, sequencing and phylogenetic analysis. The main results are as follows:1、Composition analysis of HMW-GS of Ps. junceaTwo wheat cvs CS (Chinese Spring) and Sh (Shinchunaga) with known high-molecular-weight glutenin subunits were used as references for determining subunit compositions of four Ps. juncea accessions. There were 1-5 HMW-GSs in each of the four accessions from Ps. juncea species. The HMW-GSs varied in different seeds either within or among accessions, and the electrophoretic mobility was ranged between Bx7 and Dx2.2 subunits in wheat.2. Molecular cloning and sequence comparisons of HMW-GS genes from Ps. junceaA 1.3 Kb DNA fragment was amplified from each of the seven Ps. juncea accessions PI 531826, PI 565074、PI 565065、PI 619483、PI 429801、PI 315080 and PI 75737. These nucleotide sequences were named as HMWNs1-Ns7, The DNA lengths were 1,323 bp (HMWNs1),1,320 bp (HMWNs2-6) and 1,318 bp (HMWNs7), respectively.Sequence alignment revealed that the seven HMW-GSs sequences of Ps. juncea shared higher similarity to standard y type HMW-GSs in all structural domains of Triticeae. However, novel modifications were observed in all the domains. For example, they had a longer N-terminal region with 120 amino acid residues (AA). An extra short HNRLNLQSIEIG 12-AA peptide located at 7-18 positions, and four extra glutamines (Q) located at 70,91,92 and 105 positions, respectively, were responsible for its longer N-terminal region. Their repetitive domain consist of three different repeat motif units of hexapeptide (P/LGQGQQ), tridecapeptide (GYWY/HYQTVTSP/LQQ) and tetrapeptide (ITVS). The tridecapeptide was restricted to the current HMW-GSs, and the tetrapeptide was shared by D-hordein and the current HMW-GSs. Besides, they had a distinctive C-terminal with GGAMLANK replaced standard GRDALSASQ. A stop codon (TAG) was found in each of the seven HMW-GSs at position 34 in the C-terminal, possibly suggest that they were not expressed as normal HMW-GSs.3. Evolutionary relationshipsTwo phylogenetic trees were constructed based on the AA residues of N- and C-terminals, respectively. The tree based on N-terminal residues divided all of the HMW-GSs into two clades. The seven HMW-GSs of Ps. juncea in the current study clustered with barley D-hordein, whereas the HMW-GSs of Leymus (NsXm) and the same Ps. juncea (Ns) species in previous study were not included in the same clade. In the tree of C-terminal residues, the seven HMW-GSs of current Ps. juncea formed a separated clade of their own and closely related barley D-hordein.