Properties of the primary structure of the HMW-GS from <i>Ag. intermedium</i> in comparison with those of the representatives of wheat HMW-GS.

a<p>The nucleotide number of Open Reading Frame;</p>b<p>The number of Cys.</p

MALDI-TOF-MS analysis of peptide mass fingerprint of native 1Aiy1 subunit.

<p>Peptide mass is available at <a href="http://www.expasy.org/tools/peptide-mass.html" target="_blank">http://www.expasy.org/tools/peptide-mass.html</a>.</p

SDS-PAGE (A) and Western blotting (B) analysis of HMW-GSs of <i>Ag. intermedium</i>.

<p>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 <i>Ag. intermedium</i> 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 <i>Ag. intermedium</i>, 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.</p

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

<p>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.</p

SDS-PAGE (A) and Western blotting (B) analysis of HMW-GSs from <i>Ag. intermedium</i> and bacterial expression products.

<p>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 <i>Ag. intermedium</i> same as the lane 2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087477#pone-0087477-g001" target="_blank">Figure 1</a>, 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 <i>E. coli</i> containing pET-<i>Glu-1Ai1</i>, pET-<i>Glu-1Ai2</i>, pET-<i>Glu-1Ai3</i>, pET-<i>Glu-1Ai4</i>, pET-<i>Glu-1Ai5</i> pET-<i>Glu-1Ai6</i> and pET-<i>Glu-1Ai7</i>, 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 <i>E. coli</i>, 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 <i>Ag. intermedium</i> (lane 2).</p

Phylogenetic tree of <i>Thinopyrum intermedium</i> ( = <i>Ag. intermedium</i>) and some representative HMW-GSs from <i>Triticeae</i>.

<p>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 <i>Agropyron intermedium</i> with <i>Thinopyrum intermedium</i>.</p

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 <i>Ag. intermedium</i> and several representative HMW-GSs from <i>Triticum</i> genus.

<p>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.</p