Diversity of rice glutelin polypeptides in wild species assessed by the higher‐temperature sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and subunit‐specific antibodies

Abstract

In efforts to find genetic resources with high nutritional value of rice seed, we assessed the diversity of the major storage protein glutelin in 13 wild and 2 cultivated rice species by a unique SDS-PAGE method and subunit-specific antibodies. Maximum separation of microheterogeneous glutelin alpha-polypeptides, which is a prerequisite for the diversity evaluation, could be attained by SDS-PAGE performed at higher temperature (45 degrees C) than the generally employed temperatures (4-25 degrees C). Seven antipeptide antibodies were raised against subunit-specific epitope sequences designed at five sites from four variable regions spanning the glutelin alpha-polypeptides. High specificity of each antibody was confirmed using rice glutelin mutants, and demonstrated considerable variation in amino acid sequence and accumulation level of glutelin subunit in wild species, in combination with the higher-temperature SDS-PAGE. The degree of the variation was, however, changed according to the site of variable regions and the type of subunit. Some wild species accumulated nutritious GluB subunits more than cultivated rice. The wild species Oryza longiglumis and O. brachyantha had glutelin with low reactivity against most antibodies examined in this study, reflecting the significant divergence. Such wild species may hopefully serve as important genetic resources for nutritional improvement of cultivated rice.

Glutelin is partially degraded in globulin-less mutants of rice (Oryza sativaL.)

“…Electrophoresis in the first dimension (NEPHGE) was performed at 200 V for 15 min, 300 V for 15 min, 400 V for 15 min, 500 V for 15 min, and 750 V for 5 h. Electrophoresis in second dimension was carried out by higher temperature SDS-PAGE method according to Khan et al (2008a) at constant voltage of 200 V for 80 min at 45 °C. The immunoblotting was performed according to the method mentioned in our previous report (Khan et al, 2008a). Briefly, the proteins resolved by SDS-PAGE and 2D-PAGE were electrophoretically transferred to nitrocellulose membrane.…”

“…The sections on a slide glass were treated with acetone, PBS, and blocking buffer followed by primary and secondary antibodies. The primary antibodies were 1,000-fold diluted anti-glutelin A1(No.2) antibody of mouse (Khan et al, 2008a) and 2000-fold diluted anti-alpha globulin antibody (prepared against peptide sequence LTGRERFQPMFRRPGALG in rabbit). The secondary antibodies were 500-fold diluted anti-mouse IgG conjugated FITC (green) and 2000-fold diluted anti-rabbit IgG conjugated Cy3 (red).…”

“…The above observations suggest that the spots X1 and X2 were at least in part derived from GluB4 and that the spot X1 was shortened and the spot X2 was more shortened at the C-terminus of α polypeptides. The anti-B4(No.4a) and anti-B4(No.4b) were prepared against the epitopes of 32-39 residues and 2-11 residues from the C-terminus of GluB4 α polypeptide, respectively (Khan et al, 2008a), implying that the epitope regions were degraded in the spot X2 and X1, respectively. Because the MW of X3/X4/X5 was similar to that of X2, we speculated that partial degradation occurred at the equivalent region to X2 in X3/X4/X5.…”

Capillary electrophoresis for analysis of microheterogeneous glutelin subunits in rice (Oryza sativa L.)

“…However, the quantification of each subunit (band) by conventional 1-D gel electrophoresis is difficult because of insufficient band separation. Although an SDS-PAGE method at a higher temperature has been devised to maximize the separation [5], the quantification using this method requires a specific antibody reaction. The insufficient separation of the glutelin band is largely due to the microheterogeneity of the glutelin subunits.…”

“…Previously, we characterized the major subunits of glutelin and demonstrated that all GluA and GluB4 subunits polymerize because of a specific cysteine (Cys) residue [2]. We have also raised subunitspecific antibodies to determine the subunit diversity in African rice and 18 wild species [5]. Although promising wild species were identified, large-scale screening, which is inefficient using gel electrophoresis, might be necessary for the further identification of superior germplasms.…”