Structural Characterization and Evolutionary Relationship of High-Molecular-Weight Glutenin Subunit Genes in Roegneria nakaii and Roegneria alashanica

Abstract

The Roegneria of Triticeae is a large genus including about 130 allopolyploid species. Little is known about its high-molecular-weight glutenin subunits (HMW-GSs). Here, we reported six novel HMW-GS genes from R. nakaii and R. alashanica. Sequencing indicated that Rny1, Rny3, and Ray1 possessed intact open reading frames (ORFs), whereas Rny2, Rny4, and Ray2 harbored in-frame stop codons. All of the six genes possessed a similar primary structure to known HMW-GS, while showing some unique characteristics. Their coding regions were significantly shorter than Glu-1 genes in wheat. The amino acid sequences revealed that all of the six genes were intermediate towards the y-type. The phylogenetic analysis showed that the HMW-GSs from species with St, StY, or StH genome(s) clustered in an independent clade, varying from the typical x- and y-type clusters. Thus, the Glu-1 locus in R. nakaii and R. alashanica is a very primitive glutenin locus across evolution. The six genes were phylogenetically split into two groups clustered to different clades, respectively, each of the two clades included the HMW-GSs from species with St (diploid and tetraploid species), StY, and StH genomes. Hence, it is concluded that the six Roegneria HMW-GS genes are from two St genomes undergoing slight differentiation.

Keywords: Roegneria alashanica; Roegneria nakaii; gene cloning; high-molecular-weight glutenin subunits; phylogenetic analysis; variation.

Figure 1

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (a) and Western blotting (b) patterns of the high-molecular-weight glutenin subunits (HMW-GSs) from R. nakaii and R. alashanica. The HMW-GSs in Roegneria species are indicated by arrows. The HMW-GSs (1Dx2, 1Bx7, 1By8, and 1Dy12) of common wheat variety Chinese Spring were used as controls.

Figure 2

Electrophoretic profiles of PCR products corresponding to the complete coding sequences of HMW-GS genes from R. nakaii and R. alashanica. The target bands are indicated by arrows.

Figure 3

Comparison of the deduced amino acid sequences of Rny1, Rny3, and Ray1 (a); and Rny2, Rny4, and Ray2 (b). The irregular repeat motifs are underlined, indels and frame shift region are boxed, and in-frame stop codons are marked by solid arrows.

Figure 4

Sequence alignment of the six novel Roegneria HMW-GS genes, along with the other representative HMW-GS genes. (a) Signal peptides and N-terminal domains; (b) C-terminal domains. The six Roegneria HMW-GS genes are indicated by solid triangles on the left. The deleted hexapeptide crossing signal peptide and N-terminal domain in some subunits, and an undecapeptide (LAAQLPAMCRL) in the C-terminal associated with x-type subunits in wheat are marked by boxes, and the different undecapeptides of y-type subunits in wheat are shadowed. The conserved cysteine residues in the N- and C-terminal domains are indicated by arrows. The extra glutamine residue conserved in the N-terminal of all x-type and some y-type subunits is marked by an inverted solid triangle on top of the subfigure (a).

Figure 5

Heterologous expression of the HMW-GS genes from R. nakaii and R. alashanica in E. coli. The electrophoresis patterns showed the three isopropyl β-d-1-thiogalactopyranoside (IPTG)-induced Roegneria HMW-GSs in E. coli, Rny1, Rny3, and Ray1 had the same mobility with those of HMW-GSs extracted from Roegneria seeds. The three pseudogenes, Rny2, Rny4 and Ray2, did not express protein under the IPTG-induced condition. The HMW-GSs extracted from Roegneria seeds (lanes R. nakaii GT1, GT2, and R. alashanica) and expressed in E. coli (lanes pET30a-Rny3I, pET30a-Rny1I, and pET30a-Ray1I) are marked by arrows. The Chinese Spring HMW-GSs (1Dx2, 1Dy12, 1Bx7, 1By8) extracted from seeds were used as controls.

Figure 6

Phylogenetic trees of HMW-GSs from R. nakaii and R. alashanica and their orthologous subunits from wheat and other related species. The phylogenetic trees were constructed based on the amino acid sequences of N-terminal with the first three motifs of the central repetitive domain (a) and the C-terminal with the last six motifs of the central repetitive domain (b) using maximum likelihood method. Bootstrap values ≥50% estimated based on 500 replications are shown above the branches. The six novel Roegneria subunits are marked by solid triangles.

Figure 6

Phylogenetic trees of HMW-GSs from R. nakaii and R. alashanica and their orthologous subunits from wheat and other related species. The phylogenetic trees were constructed based on the amino acid sequences of N-terminal with the first three motifs of the central repetitive domain (a) and the C-terminal with the last six motifs of the central repetitive domain (b) using maximum likelihood method. Bootstrap values ≥50% estimated based on 500 replications are shown above the branches. The six novel Roegneria subunits are marked by solid triangles.