Identification and characterization of high-molecular-weight glutenin subunits from Agropyron intermedium

Abstract

High-molecular-weight glutenin subunit (HMW-GS) is a primary determinant of processing quality of wheat. Considerable progress has been made in understanding the structure, function and genetic regulation of HMW-GS in wheat and some of its related species, but less is known about their orthologs in Agropyron intermedium, a useful related species for wheat improvement. Here seven HMW-GSs in Ag. intermedium were identified using SDS-PAGE and Western blotting experiments. Subsequently, the seven genes (Glu-1Aix1 ∼ 4 and Glu-1Aiy1 ∼ 3) encoding the seven HMW-GSs were isolated using PCR technique with degenerate primers, and confirmed by bacterial expression and Western blotting. Sequence analysis indicated that the seven Ag. intermedium HMW-GSs shared high similarity in primary structure to those of wheat, but four of the seven subunits were unusually small compared to the representatives of HMW-GS from wheat and two of them possessed extra cysteine residues. The alignment and clustering analysis of deduced amino acid sequences revealed that 1Aix1 and 1Aiy1 subunits had special molecular structure, belonging to the hybrid type compounding between typical x- and y-type subunit. The xy-type subunit 1Aix1 is composed of the N-terminal of x-type and C-terminal of y-type, whereas yx-type subunit 1Aiy1 comprises the N-terminal of y-type and C-terminal of x-type. This result strongly supported the hypothesis of unequal crossover mechanism that might generate the novel coding sequence for the hybrid type of HMW-GSs. In addition to the aforementioned, the other novel characteristics of the seven subunits were also discussed. Finally, phylogenetic analysis based on HMW-GS genes was carried out and provided new insights into the evolutionary biology of Ag. intermedium.

Figure 1. SDS-PAGE (A) and Western blotting (B) analysis of HMW-GSs of Ag. intermedium.

Lane 1 shows the named HMW-GSs from common wheat variety Chinese Spring as a control. Lanes 2∼7 show the HMW-GSs from six representative seeds of the Ag. intermedium line used in this study. The seven expressed HMW-GSs with distinct electrophoretic mobility comparing with Chinese Spring were detected by SDS-PAGE (A) and were confirmed using Western blotting experiment with polyclonal antibody specific for HMW-GSs (B). Among the seven HMW-GSs from Ag. intermedium, three subunits (marked with solid triangles in lane 2 of B) share comparable electrophoretic mobility with Chinese Spring, the other four subunits (marked with hollow triangles in lane 2 of B) moved faster than those HMW-GSs from Chinese Spring.

Figure 2. Amplification of complete ORFs coding for HMW-GSs of Ag. intermedium.

Lane 1 is DNA marker III from TIANGEN and lane 2∼7 shows the PCR bands from Ag. intermedium seedlings corresponding to the seeds of lane 2∼7 in Figure 1.

Figure 3. SDS-PAGE (A) and Western blotting (B) analysis of HMW-GSs from Ag. intermedium and bacterial expression products.

Lane 1 is HMW-GSs from common wheat variety Chinese Spring, the four expressed HMW-GSs are noted on left; Lane 2 is native HMW-GS from the seed of Ag. intermedium same as the lane 2 in Figure 1, the seven expressed HMW-GSs are marked with triangles; Lane 3, 5, 7, 9, 11, 13 and 15 are total cell proteins from IPTG induced E. coli containing pET-Glu-1Ai1, pET-Glu-1Ai2, pET-Glu-1Ai3, pET-Glu-1Ai4, pET-Glu-1Ai5 pET-Glu-1Ai6 and pET-Glu-1Ai7, respectively, whereas the dextral lanes for each of them shows the total cell proteins from their bacterial cells without induction of IPTG. The seven expressed target proteins in E. coli, which were detected by SDS-PAGE (marked with arrows in A) and were confirmed by Western blotting (lanes 3, 5, 7, 9, 11, 13 and 15 in B), share comparable electrophoretic mobility with those native HMW-GSs from Ag. intermedium (lane 2).

Figure 4. Alignments and clustering analyses based on N-terminal (A), C-terminal (B) and the last 93 residues in the repetitive region (C) of the HMW-GSs from Ag. intermedium and several representative HMW-GSs from Triticum genus.

Noticeably, the subunit 1Aix1 possesses a N-terminal clustered to x-type subunits (A) and a C-terminal and last part of repetitive region clustered to y-type subunits (B and C). Conversely, the subunit 1Aiy1 possesses a N-terminal more similar to y-type subunits (A) and a C-terminal and last part of repetitive region more similar to x-type subunits (B and C). The default parameters were used for full alignment and clustering analysis of sequences by aid of DNAMAN version 5. 2. 2.

Figure 5. Phylogenetic tree of Thinopyrum intermedium ( = Ag. intermedium) and some representative HMW-GSs from Triticeae.

This phylogenetic tree was constructed with Maximum Likelihood Estimation method based on the nucleotide sequences encoding signal peptide and N-terminal conserved region of HMW-GSs plus the next three repeat units, one dodecapeptide, one undecapeptide and one hexapeptide repeat. D-hordein from barley was used as outgroup. The species names of HMW-GS genes in this figure are consistent with their accession names in GenBank, so here we replaced Agropyron intermedium with Thinopyrum intermedium.

Figure 6. Illustration for the developmental mechanism of two hybrid HMW-GSs based on unequal double crossover hypothesis.

The broken line box indicates the double crossover region. The xy and yx represent the hybrid subunit with 5′region of x-type and 3′region of y-type and the hybrid subunit with 5′region of y-type and 3′region of x-type, respectively.