Identification Of Active Y-type Glu-A1Genes From Triticum Dicoccoides

The high molecular weight glutenin subunit (HMW-GS), one of the most important storage proteins in wheat, of which contents and compositions play a key role in determining the end-use quality of wheat flours. In common wheat, gene coding for subunit1Ay is unexpressed. However, this subunit is active in many lines of diploid wheat (AA), and tetraploid wheat with the genomes AABB and AAGG, and hexaploid wheat (AAAAGG). In this study, the characterization of1Ay gene of wild emmer wheat (T. dicoccoides, AABB) were firstly described by using SDS-PAGE, gene cloning, sequencing, bacterial expression and cluster analyses. The main results are as follows.1. HMW glutenin subunits were separated by SDS-PAGE, which were extracted by two kinds of methods, general method and selective precipitating. Four HMW-GS bands were detected in the lines D141and D129of T. dicoccoides. Their lAy subunits had similar electrophoretic mobility, which was faster than1Dy12in common wheat.2. Four DNA bands were produced by PCR from the wild emmer lines D141and D129using the special primers of HMW-GS genes. The smallest DNA fragments about1.8Kb and1.7Kb were similar to the published lAy subunit. Their full lengths of open reading frame (ORF) were1830bp and1671bp, which have been deposited in GenBank database with the accessions No. JF519636and No. JF519635, respectively. And, the JF519635was the smallest1Ay gene, so far, encoding555amino acid residues. Blast analysis indicated that they were belonged to the gene encoding y-type glutenin subunit at Glu-A1locus due to the highest identities of98%and96%to the published active lAy sequences, respectively. They had similar trend of amino acid variations in repeat consensus to those of all the published active lAy subunits, which occurred more at the positions1and4in hexapeptides, and2,5and7in nonapeptides. And, JF519636has the highest variations accounting for1.197in the repetitive consensuses. They have16and17unique single point mutations (SPMs), respectively, compared with previously reported active1Ay subunits.3. The secondary protein structures were predicted using PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipredd/). The coils accounted for93.26%and90.63%, and β-strands were absent in the JF519636and JF519635, respectively.4. The authenticity of the cloned1Ay gene of JF519636and JF519635was confirmed by successful expression of the encoding regions in E. coli. The expressed mature protein showed a eletrophoretic band identical to the lAy from seed of T. dicoccoides lines D141and D129.5. Phylogenetic tree consisting of22HMW glutenin subunits including the present two1Ay subunits and other20previously published HMW-GSs was constructed. The present lAy subunits JF519636and JF519635were closely clustered together, and, more closely with four lAy subunits (EU984503, FJ404595, AY245578and AM183223) from5published1Ay nucleotide sequences in T. urartu. This intensified the viewpoint that T. urartu provides the donor A genome in T. dicoccoides.