**6. High‐pressure liquid chromatography (HPLC) analysis of total amino acid content**

An important feature of transgenic plants of sorghum and maize with silencing of prolamin genes is an increased proportion of essential amino acids in kernels, in particular the lysine proportion. Previously, this effect was observed in the silencing of genes of the main prolamin fractions: α‐zein [22, 23] and α‐kafirin [24, 27]. It was assumed that the suppression of the synthesis of these proteins, characterized by a low content of lysine, results in upregulating non‐storage protein genes and appearance of lysine‐rich proteins [23, 27].

In our experiments, the total amino acid content in the kernels of three transgenic plants from the T2 generation with high *in vitro* protein digestibility: #94‐2‐11, #94‐2‐04 (both with modi‐ fied endosperm) and #94‐3‐08 (with vitreous endosperm) was studied by using HPLC [28]. As can be seen from **Table 4**, content of a number of amino acids (leucine, proline, serine, isoleu‐ cine, histidine, tyrosine) and total amino acid content were significantly reduced in transgenic plants #94‐2‐04 (−40.2%, in comparison with the original non‐transgenic line) and #94‐3‐08 (−22.8%). At the same time, the relative content of two major essential amino acids, lysine and threonine, significantly increased. Lysine proportion is increased by 1.6–1.7 times: from 1.54% of total amino acid content in the flour of original non‐transgenic line Zh10 to 2.41–2.63% in transgenic plants #94‐3‐08 and #94‐2‐04, respectively, with vitreous and modified endosperms.

Development of Transgenic Sorghum Plants with Improved *In Vitro* Kafirin Digestibility http://dx.doi.org/10.5772/intechopen.69973 107


Thus, the comparison of electrophoretic spectra of endosperm proteins before and after pep‐ sin treatment showed a high level of kafirin digestibility in transgenic sorghum plants, har‐ boring genetic construct for silencing of the γ‐kafirin gene. Such electrophoretic spectra of digested endosperm proteins are not characteristic of ordinary sorghum cultivars obtained by classical breeding [40–42] except highly digestible sorghum mutant (*hdhl*) and its hybrids [37–39]. Apparently, a decrease in the level of γ‐kafirin increases the digestibility of α‐kafirins. This increase may be due to chemical reasons (reduction of polymerization) and/or physical reasons (change in the spatial arrangement of α‐kafirins in the protein bodies that increase

Earlier it was reported on obtaining of transgenic sorghum plants carrying genetic constructs for silencing of γ‐ and α‐kafirins, which were characterized by increased *in vitro* protein digestibility [25–27]. However, electrophoretic spectra of endosperm proteins after pepsin treatment were not shown in these studies. It should be noted also that in these studies improvement of kafirin digestibility was induced by complex genetic constructs that contained inverted repeats of sev‐ eral kafirin genes (δ2, γ1, γ2; or α1, δ2, γ1, γ2). These repeats were separated by the sequence of ADH1 intron, and the constructs were driven by the maize 19‐kDa α‐zein promoter [24–26]. In another work [27], the genetic construct included the complete sequence of the γ‐kafirin gene, which was terminated by a nucleotide sequence of the self‐cleaving ribozyme of tobacco ringspot virus that should destroy γ‐kafirin mRNA. In our study [28], the effect was achieved by using a simpler genetic construct, containing inverted repeats of a short segment of the gene γ‐kafirin (307 bp) separated by *ubi1*‐intron gene, under the control of the constitutive *35S*‐promoter, which

**6. High‐pressure liquid chromatography (HPLC) analysis of total amino** 

An important feature of transgenic plants of sorghum and maize with silencing of prolamin genes is an increased proportion of essential amino acids in kernels, in particular the lysine proportion. Previously, this effect was observed in the silencing of genes of the main prolamin fractions: α‐zein [22, 23] and α‐kafirin [24, 27]. It was assumed that the suppression of the synthesis of these proteins, characterized by a low content of lysine, results in upregulating

In our experiments, the total amino acid content in the kernels of three transgenic plants from

 generation with high *in vitro* protein digestibility: #94‐2‐11, #94‐2‐04 (both with modi‐ fied endosperm) and #94‐3‐08 (with vitreous endosperm) was studied by using HPLC [28]. As can be seen from **Table 4**, content of a number of amino acids (leucine, proline, serine, isoleu‐ cine, histidine, tyrosine) and total amino acid content were significantly reduced in transgenic plants #94‐2‐04 (−40.2%, in comparison with the original non‐transgenic line) and #94‐3‐08 (−22.8%). At the same time, the relative content of two major essential amino acids, lysine and threonine, significantly increased. Lysine proportion is increased by 1.6–1.7 times: from 1.54% of total amino acid content in the flour of original non‐transgenic line Zh10 to 2.41–2.63% in transgenic plants #94‐3‐08 and #94‐2‐04, respectively, with vitreous and modified endosperms.

allowed us to reach apparently rather high level of silencing of a target gene.

non‐storage protein genes and appearance of lysine‐rich proteins [23, 27].

their availability to pepsin digestion).

**acid content**

106 Plant Engineering

the T2

Notes: Values are mean ± standard error from three replications. Data marked in bold differ significantly from the original non‐transgenic (control) line Zh10 at *P* < 0.05 (\*) and *P* < 0.01 (\*\*) according to Student's T‐test. Percentage of reduction is indicated in parenthesis.

Data for total amount of amino acid content and for percentage of lysine and threonine from the total amino acid content followed by the same letter did not differ significantly (*P* < 0.05) according to Duncan Multiple Range Test.

**Table 4.** Total amino acid content in kernels of transgenic sorghum plants obtained by genetic transformation with *A. tumefaciens* GV3101/pNRKAFSIL (g/100 g flour) (published with the permission of the publishing house "Nauka").

Such increase of the relative content of lysine and threonine in transgenic sorghum plants, coupled with a significant reduction of the total level of amino acids (**Table 4**), presumably was caused by decrease in the content of α‐kafirins poor in lysine and threonine, whereas the synthesis of other proteins remained undisturbed. Accordingly, the relative proportions of lysine and threonine increased. The lower α‐kafirins content in transgenic plants relative to the original non‐transgenic line is clearly evident in the above electrophoresis photographs (**Figures 8** and **9**). Perhaps the suppression of the synthesis of γ‐kafirin disrupts the formation of protein bodies and prevents the accumulation of α‐kafirins, but does not affect the synthe‐ sis of other proteins richer in lysine and threonine.
