Characterization And Isolation Of High Molecular Weight Glutenin Subunits Genes From Crithopsis Delileana

Crithopsis delileana (Schult) Roshev (2n=2x=14, KK) is the only member of the genus Crithopsis Jaub. Et Spach. in the Triticeae group. The classification and evolution about this species had been extensively investigated, whereas little was known about the HMW glutenin of this species. For the better understanding of HMW glutenin of C. delileana, the HMW glutenin subunits composition and its coding genes in C. delileana were investigated.1. HMW glutenin subunit composition analysis of C. delileanaIn SDS-PAGE analysis by general extraction method of seed protein, two candidate HMW glutenin subunits were detected in 2 accessions of C.delileana from Demark. One putative HMW glutenin subunit has the similar electrophoresis mobility between subunits By8 and By 10 in hexaploid wheat, and the other band has abnormal faster electrophoresis mobility than any of the y-type glutenin in wheat. Using acetone extraction method, it was found that only one band with electrophoresis mobility between subunits By8 and By 10 in hexaploid wheat was detected.2. Molecular cloning of Kx and Ky subunit genes from C. delileanaOwing to that no HMW glutenin subunits variation was detected in C.delileana accessions, one accession was selected for the further studying. In genomic PCR reactions using the primer pairs of P1 and P2, two fragments (2.1kb and 1.7kb) were specifically amplified. It was confirmed that the two fragments were the coding genes of subunit Kx and Ky (not expressed) by sequence analysis. The sequences of HMW glutenin genes Kx and Ky had been deposited in the GenBank under accession numbers AY804128 and AY834230, respectively.The full coding region of Kx was 2,046bp, and could encode a mature protein with 661 amino acids residues. The coding sequence of Kx has the typical primary structures of HMW glutenin subunits. In the deduced amino acid sequence of Kx, a conserved signal peptide with 21 residues, a N-terminal region with 86 residues, acentral repetitive domain with 533 residues and a C-terminal region with 42 resisdues were observed. The repetitive domain consists of interspersed repeats based on 9 tripeptide (consensus GQQ), 56 hexapeptide (consensus PGQGQQ) and 16 nonapeptide (consensus GYYPTSPQQ) motifs. Two longer repeat units (11 followed by 12 residues) and a tripeptide (GYD) were adjacent to each other at the beginning and end of the repetitive central domain, respectively. It was indicated that the amino acid sequence of Kx were different from the HMW glutenin representatives by substitutions, insertions and/or deletions involving single amino acid residues or motifs. The size of the repetitive domain of the Kx was substantially smaller than those of the known subunits.The coding sequence of HMW glutenin subunit Ky was 1,725 bp, and had the similar typical primary structure to those of known y-type HMW glutenin. Due to an in-frame stop codon in the coding regions at position 1558 bp, the HMW glutenin Ky could not to be expressed as an active mature protein. It was in agreement with SDS-PAGE analysis, and gave the further support to the SDS-PAGE analysis results that there was only one expressed HMW glutenin subunit in C.delileana3. Expression of the coding sequence of HMW glutenin gene Kx in E.coliThe HMW glutnein subunit gene Kx has been successfully expressed in E.coli. The expressed HMW glutenin subunit of cloned gene in E.coli had the same mobilities to those of seeds in SDS-PAGE.4. Evolutionary relationshipsIn the phylogenetic relationship analysis, N -terminal amino acid sequences were used to construct a dendrogram of the Kx subunit and Ky subunit (not expressed) with representives HMW subunits from A, B and D genomes of wheat, C and U genomes of Aegilops, the R genome of S. cereal and the H genome of barley using the corresponding region of barley D-hordein as an outgroup. On the resulting dendrogram, it was obvious that the Kx subunit could be clustered together with Ax1 subunit by an interior parallelled branch, and the Ky subunit (not expressed) was most closely related to the Ay subunit. It was suggested that the HMW glutenins subunits from C. delileana were most closely related to those of subunits encoded by the A genome of wheat.