ZEIN CODING SEQUENCE ANALYSES FOR MAIZE GENOTYPING AND ZEIN PROTEIN MANIPULATION TOWARDS THE IMPROVEMENT OF THE MAIZE SEED PROTEIN QUALITY

Maize (Zea mays) is an important source of proteins for human and animal nutrition. However, because of the lack of lysine and the low content in methionine and tryptophan, maize’s proteins are of low quality. These deficiencies mainly result from the low levels of these essential amino acids in the zein storage proteins, which account for 50% of the total protein in mature seed. In this context, the first aim of this PhD thesis has been to develop artificial zein genes encoding for polypeptides with a higher content in lysine and methionine, and capable to be sorted and correctly accumulated into the endosperm, as occur for natural zein polypeptides. Two strategies have been employed for maize bio-fortification. First, we exploited the natural heterogeneity among α-zein genes to create a synthetic gene, ZRK, in which six arginine residues have been substituted with lysine. Then, by combining the N-terminal methionine-rich G3 sequence and the C-terminal lysine-rich region of Histone3 and Histone4 of maize, the G3H3 and G3H4 artificial genes were created, respectively. In vitro and in vivo expression analyses of these genes showed that all synthetic proteins are synthesized and accumulated into the ER membranes of either the rabbit reticulocyte/canine membrane system or of transformed tobacco protoplasts. The second aim of this thesis has been to use the wide heterogeneity of zein gene family to obtain an intra-species recognition tool, or individual barcode, for inbreds and Lombard varieties discrimination. Lombard varieties and maize inbreds were analysed by 2D gel protein fractionations and DNA gel blot analyses. For each genotype the 2D and Southern blot pattern were converted into a binary code, and then into a barcode. In both the approaches, each genotype was univocally identified making zeins a valuable tool for identification of maize germplasm.