Abstract
BackgroundOne of the goals of plant breeding is to create crops to provide better nutrition for humans and livestock. Insufficient intake of protein is one of the most severe factors affecting the growth and development of children in developing countries. More than a century ago, in 1896, Hopkins initiated the well-known Illinois long-term selection for maize seed protein concentration, yielding four protein strains. By continuously accumulating QTLs, Illinois High Protein (IHP) reached a protein level 2.5-fold higher than normal maize, with the most increased fraction being the zein protein, which was shown to contain no lysine soon after the long-term selection program initiated. Therefore, IHP is of little value for feeding humans and monogastric animals. Although high-lysine lines of non-vitreous mutants were based on reduced zeins, the kernel soft texture precluded their practical use. Kernel hardness in opaque 2 (o2) could be restored in quality protein maize (QPM) with quantitative trait loci called o2 modifiers (Mo2s), but those did not increase total protein levels.MethodsThe most predominant zeins are the 22- and 19-kDa α-zeins. To achieve a combination of desired traits, we used RNA interference (RNAi) against both α-zeins in IHP and evaluated the silencing effect by SDS-PAGE. Total protein, amino acid composition and kernel texture were analyzed.ConclusionsThe α-zeins were dramatically reduced, but the high total seed protein level remained unchanged by complementary increase of non-zein proteins. Moreover, the residual zein levels still allowed for a vitreous hard seed. Such dramatic rebalancing of the nitrogen sink could have a major impact in world food supply.
Highlights
Maize (Zea mays), commonly known as corn, produces the highest yield among the major crops in the world, providing food for humans and feed for livestock
Introgressed QTLs are capable of raising the protein concentration in Illinois High Protein (IHP) more than twice that in normal maize, with the most increased fraction being the alcohol-soluble proteins or prolamins
Despite its higher protein content, IHP is of little value for feeding monogastric animals
Summary
Genetic Stocks The P6z1RNAi transgenic plant has been described previously [13]. The P6z1RNAi transgenic plant was backcrossed to B73 for two generations (Fig. 3A), which consistently showed 1:1 ratio of vitreous (non-green) and opaque (green) seeds segregating in each generation. Total Zein and Non-zein Protein Extraction, Protein and Amino Acid Composition Analysis. The dry kernels were wrapped individually in two layers of thick aluminium foil and crushed into fine flour by a heavy hammer. Solids remaining in the tube were resuspended with zein extraction buffer to completely remove the zeins from other proteins. This step was repeated for 3 times. The residual solids were suspended in 400 ml of non-zein extraction buffer (12.5 mM sodium borate, 5% SDS and 2% 2-mercaptoethanol (vol/vol)). The mixture was centrifuged at 13,000 rpm for 10 min, and 100 ml of the non-zein supernatant was transferred to a new tube. Kernels were truncated and scoped under incandescent and fluorescent light dissection microscopes, respectively (WILD M3 and Leica MZ16 F)
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