Aggregation of lysine-containing zeins into protein bodies in Xenopus oocytes

Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and tryptophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.     

Resistance of soybean vegetative storage proteins (S-VSPs) to proteolysis by rumen microorganisms

Soybean vegetative storage proteins (S-VSPs) are lysine-rich and, hence, are potentially of high nutritive value for high productive ruminants. Using S-VSPs from wild-type soybean and from transgenic tobacco plants expressing either one of the two S-VSPs subunits (S-VSP alpha or S-VSP beta) or both, we tested their stability in cow rumen fluid under in situ conditions, using SDS-polyacrylamide gel electrophoresis. Proteolysis and degradation pattern of S-VSPs from transgenic tobacco leaves occurred relatively fast compared with that of wild-type (WT) soybean plants. Comparing the two S-VSPs subunits expressed in transgenic plants, we found that S-VSP alpha was degraded much faster than S-VSP beta. The degradation pattern of S-VSPs in transgenic tobacco plants expressing both subunits resembled that of WT soybean. In contrast, the degradation pattern of transgenic tobacco plants expressing a single subunit was different. These finding suggest that the quaternary structure of S-VSPs may be an important factor determining their resistance to rumen degradation. Our results also suggest that the stability to rumen proteolysis of a given protein, when expressed in a transgenic plant, may not always be predictable and has to be verified.     

Genotype-by-environment effects on the performance of recombinant inbred lines of Virginia-type peanut

Peanut (Arachis hypogaea L.) is known to be sensitive to genotype-by-environment interaction (GEI) effects. While previous studies have reported strong GEI effects on peanut yield, most of those studies involved a relatively small number of unrelated genotypes. We examined the extent of GEI effects in elite Virginia-type peanut using a large population of recombinant inbreed lines (RILs). Two-hundred-sixty-six F₇ RILs derived from different cultivars were grown in three environments. Net pod yield (NPY) was evaluated along with 11 other traits. ANOVA revealed that genotype and environment affected all of the examined traits, except for the triplet trait. The substantial influence of the environment was also demonstrated in a genetic-parameter analysis, in which the phenotypic variation coefficients were almost double those for genotypic variation. Still, relatively high heritability and genetic gain values were found for pod weight and NPY. Since NPY is the main target for selection, it was analyzed further. Path analysis showed that NPY is most directly influenced by pod weight and the shelling ratio. A significant GEI effect on NPY was identified using an AMMI model that described 42.7% of the total variation. This GEI component was subjected to a principal components analysis, which confirmed that the variability due to the different environments was greater than the variability that could be attributed to the different genotypes. Yet, several lines had stable yields across environments. These results demonstrate the importance of multi-location phenotyping for QTL analyses and crop improvement in peanut.