Molecular characterization of Zea mays glutelin genes and
--B. Lazzari, F. Sparvoli, L. Bernard and A. Viotti
Zeins and glutelins are the most important storage proteins of maize endosperm. All the polypeptides belonging to these two families have a signal peptide which allows them to enter the E.R. (endoplasmic reticulum). Once there, these proteins are stored in enlargements of the E.R., typical of maize endosperm, called protein bodies (P.B.).
The glutelin family is mainly composed of four polypeptides. Two of them, G1-204 and G1-164, are cysteine-rich (6.6%) and are closely related to each other (Prat et al., Gene 52:41-49). The genes of this subfamily code respectively for polypeptides of 204 and 164 amino acids and share a high degree of homology, except for the fact that G1-204 contains eight repeats of the hexapeptide "PPPVHL", which are partially deleted in G1-164. The presence in G1-204 of repeated proline triplets decreases the protein migration in SDS-PAGE, so that the polypeptide has an apparent molecular weight of 28Kd, while its real molecular weight is 22.8Kd. G1-164 migrates with an apparent molecular weight of 16Kd. The high degree of homology of G1-164 and G1-204 suggests a common origin of the two genes, which could have diverged during evolution from a unique progenitor.
The other two glutelin genes, called G2 and G3, are methionine-rich (11% and 20%, respectively) and code for polypeptides of apparent molecular weight of 15 and 10Kd. These polypeptides are also known as b and s zeins (Pedersen et al., J. Biol. Chem. 261: 6279-6284; Anderson, Kirihara et al., Mol. Gen. Genet. 211: 477-484).
The complete cDNA sequences of the four genes have been amplified by PCR from total endosperm RNA using specific primers and have been used as probes in Southern blotting experiments. Total DNA extracted from different maize lines (W64A, A69Y and W22) and tissues and digested with different restriction enzymes has been loaded on agarose gels and blotted to Hybond N+ membranes.
Hybridization revealed that there is no difference concerning G1, G2 and G3 patterns between 2n and 3n tissues (embryo and endosperm, respectively), while there is polymorphism in the different maize lines. Southern blotting analysis has also been performed to determine the number of copies per haploid genome of these genes. As G1-204 and G1-164 cross-hybridize, due to their high homology, their copy number has been determined using only the 3' end as a probe, which is almost identical in the two genes: data show that there are between 5 and 10 copies per haploid genome of genes belonging to the G1 subfamily (G1-204 + G1-164).
The same experiment has been carried out with G2 and G3 probes: G2 seems to be present in 1-2 copies, and G3 is present in 2-3 copies per haploid genome.
The importance of glutelins as storage proteins is mainly due to their high sulphur content, especially if we consider that zein, which is the most important family of storage proteins in maize, has a very low content of methionine and cysteine.
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