**3. Analysis of electrophoretic spectra of endosperm proteins in plants with genetic construct for RNA silencing of the γ‐kafirin gene**

To identify the expression of the introduced genetic construct, the experiments on SDS‐PAGE of endosperm proteins were performed. The samples (20 mg of flour) were incubated with a sample buffer (0.0625 M Tris·HCl, pH 6.8) under reducing conditions (2% SDS, 5% β‐mer‐ captoethanol, destroying the S–S bonds of kafirin polymers) or in native, non‐reducing con‐ ditions (without β‐mercaptoethanol) at 100°C for 90 s. The samples were centrifuged, and supernatant was used for SDS‐PAGE in 13.0% (w/v) polyacrylamide gel (PAG) according to modified Laemmli method [35]. The gels were stained with Coomassie Brilliant Blue R‐250. The electrophoretic spectra were carefully studied, and particular attention was paid to the γ‐kafirin content, the suppression of which was to be expected, and to content of kafirin oligo‐ mers (≈47 and ≈66 kDa), which consist from α‐ and γ‐kafirins [14, 17, 18].

It was found that in kernels of original non‐transgenic line Zh10 content of polypeptides with Mr ≈47 and ≈66 kDa was markedly higher than in transgenic plants. These differences were observed both in SDS‐PAGE performed in non‐reducing conditions (**Figure 6**) and in reduc‐ ing conditions (see Section 5).

Notably, electrophoresis in non‐reducing conditions revealed that the level of polypeptide corresponding to γ‐kafirin (28 kDa, marked by an arrow) in transgenic plants was signifi‐ cantly lowered compared to the original non‐transgenic line, which was to be expected with the silencing of the γ‐kafirin gene. In addition, as we found previously in experiments on SDS‐PAGE in reducing conditions, content of α‐kafirin monomers (25 and 23 kDa) was also

**Figure 6.** SDS‐PAGE of endosperm proteins of transgenic sorghum plants with genetic construct for silencing of the γ‐kafirin gene in non‐reducing conditions. γ‐kafirin is marked by arrow; the proteins, the amount of which varies in the original line and in transgenic plants, are marked by asterisks.

reduced in transgenic plants ([28], see Section 5). Perhaps the suppression of the synthesis of γ‐kafirin caused also effect on synthesis or accumulation of α‐kafirins.

**3. Analysis of electrophoretic spectra of endosperm proteins in plants** 

To identify the expression of the introduced genetic construct, the experiments on SDS‐PAGE of endosperm proteins were performed. The samples (20 mg of flour) were incubated with a sample buffer (0.0625 M Tris·HCl, pH 6.8) under reducing conditions (2% SDS, 5% β‐mer‐ captoethanol, destroying the S–S bonds of kafirin polymers) or in native, non‐reducing con‐ ditions (without β‐mercaptoethanol) at 100°C for 90 s. The samples were centrifuged, and supernatant was used for SDS‐PAGE in 13.0% (w/v) polyacrylamide gel (PAG) according to modified Laemmli method [35]. The gels were stained with Coomassie Brilliant Blue R‐250. The electrophoretic spectra were carefully studied, and particular attention was paid to the γ‐kafirin content, the suppression of which was to be expected, and to content of kafirin oligo‐

It was found that in kernels of original non‐transgenic line Zh10 content of polypeptides with Mr ≈47 and ≈66 kDa was markedly higher than in transgenic plants. These differences were observed both in SDS‐PAGE performed in non‐reducing conditions (**Figure 6**) and in reduc‐

Notably, electrophoresis in non‐reducing conditions revealed that the level of polypeptide corresponding to γ‐kafirin (28 kDa, marked by an arrow) in transgenic plants was signifi‐ cantly lowered compared to the original non‐transgenic line, which was to be expected with the silencing of the γ‐kafirin gene. In addition, as we found previously in experiments on SDS‐PAGE in reducing conditions, content of α‐kafirin monomers (25 and 23 kDa) was also

**Figure 6.** SDS‐PAGE of endosperm proteins of transgenic sorghum plants with genetic construct for silencing of the γ‐kafirin gene in non‐reducing conditions. γ‐kafirin is marked by arrow; the proteins, the amount of which varies in the

original line and in transgenic plants, are marked by asterisks.

**with genetic construct for RNA silencing of the γ‐kafirin gene**

mers (≈47 and ≈66 kDa), which consist from α‐ and γ‐kafirins [14, 17, 18].

ing conditions (see Section 5).

98 Plant Engineering

Noteworthy, lowered amount of protein in the lanes of transgenic plants (**Figure 6**) is not an artifact, since in each sample the same amount of flour was taken in the study; all samples were subjected to the same treatment, and the same amount of extract was taken when carry‐ ing out SDS‐PAGE. Therefore, such reduced protein content is due to the genetic character‐ istics of the samples. A similar decrease in protein content was observed in transgenic maize plants carrying constructs for RNA silencing of γ‐zein [36].
