Part of the book: Gel Electrophoresis
Improvement of nutritional value of crops is one of the main goals of plant biotechnology. These studies are extremely important for sorghum—a unique drought‐tolerant cereal crop that is of special importance for sustainable grain production in the arid regions. The major cause of relatively low nutritive value of sorghum grain is the resistance of one of its seed storage proteins, γ‐kafirin, to protease digestion. Using Agrobacterium‐mediated genetic transformation, we have obtained transgenic sorghum plants harboring a genetic construct for RNA interference (RNAi) silencing of the γ‐kafirin gene. In T1 generation, transgenic plants with modified endosperm texture were found. These plants had lowered level of the 28‐kDa γ‐kafirin protein and kafirin oligomers, which are formed by natural kafirin polymerization. In vitro protein digestibility analysis showed that the amount of undigested protein in transgenic plants was reduced by 2.9–3.2 times, in comparison with the original line, the digestibility index reached 85–88% (60% in the original line). HPLC analysis showed that total amino acid content in transgenic plants was reduced, while the lysine proportion was increased by 1.6–1.7 times. PCR analysis confirmed inheritance of the genetic construct up to T4 generation.
Part of the book: Plant Engineering
Modification of the composition of grain storage proteins is an intensively developing area of plant biotechnology, which is of particular importance for sorghum – high-yielding drought tolerant crop. Compared to other cereals, the majority of sorghum cultivars and hybrids are characterized by reduced nutritional value that is caused by a low content of essential amino acids in the seed storage proteins (kafirins), and resistance of kafirins to protease digestion. RNA interference (RNAi) by suppressing synthesis of individual kafirin subclasses may be an effective approach to solve this problem. In this chapter, we review published reports on RNAi silencing of the kafirin-encoding genes. In addition, we present new experimental data on phenotypic effects of RNAi-silencing of γ-KAFIRIN-1 gene in sorghum cv. Avans. To obtain RNAi mutants with γ-KAFIRIN-1 gene silencing we used Agrobacterium-mediated genetic transformation. Transgenic kernels had modified endosperm type with reduced vitreous layer and significantly improved in vitro protein digestibility (93% vs. 57%, according to the densitometry of SDS-PAGE patterns). SDS-PAGE of transgenic kernels showed lowered level of kafirins and appearance of globulin proteins, which were not observed in the original cultivar. For the first time, the cases of instability of inserted genetic construct were identified: elimination of ubi1-intron that is a constituent part of the genetic construct for RNAi silencing, or nos-promotor governing expression of the marker gene (bar) (in the RNAi mutants of cv. Zheltozernoe 10). The research findings presented in this chapter provide strong evidence that RNA interference can be used for improvement of the nutritional properties of sorghum grain.
Part of the book: Grain and Seed Proteins Functionality