The b-32 protein is not encoded by the opaque-6 locus

--P. Ajmone Marsan, F. Salamini, P. Franceschini, G. Monfredini and M. Motto

Several mutations affecting the accumulation of maize seed storage proteins have been described (see Motto et al., Oxford Surv. Plant Mol. Cell. Biol. 6:87-114, 1989). Phenotypically these mutant kernels are easily recognized since they are soft and opaque in contrast to wildtype kernels that have hard, translucent endosperm.

One of these mutations, opaque-2 (o2), causes a partial to nearly complete elimination of the 22kDa class of zein proteins. The o2 mutant is also unable to synthesize a number of non-zein polypeptides present in the wildtype endosperm, the most abundant of which is a 32kDa cytosolic albumin, termed b-32. Its expression during kernel development is temporarily and quantitatively coordinated with the deposition of storage proteins in endosperm tissue (Soave et al., Cell 27:403-410, 1991). The gene encoding the b-32 protein has been cloned and the complete amino acid sequence of this protein derived (Hartings et al., Plant Mol. Biol. 14:1031-1040, 1990). Furthermore, b-32 was shown to be a functional ribosome-inactivating protein and to interact with the translation machinery of the maize endosperm cell in enhancing zein synthesis (Maddaloni et al., J. Genet. Breed., in press, 1991). b-32 is coded by, or tightly linked to, the opaque-6 (O6) locus (Soave et al., Cell 27:403-410, 1981; Di Fonzo et al., Planta 167:587-594, 1986).

We have analysed the segregation pattern of a restriction fragment length polymorphism through Southern blots in seedlings derived from normal and o6 kernels. In the case of identity of the two loci a perfect cosegregation was expected. Seedlings derived from kernels of B37 inbred line and from vitreous (O6/O6 or O6/o6) and opaque (o6/o6) kernels of F2 ears in a B37 background were the sources of DNAs. A b-32 DNA insert derived from the full-length cDNA clone b-32.66 (Hartings et al., Plant Mol. Biol. 14;1031-1040, 1990) was used as a probe.

Southern hybridization of the b-32 probe to BamHI digests of genomic DNA extracted from seedlings of B37 wildtype and opaque kernels revealed restriction fragment length polymorphisms. Thirty-five DNAs from the opaque seeds (o6/o6) were further analysed and two of them showed recombination between O6 and b-32 loci. Thirty-two DNAs from the vitreous seeds (o6/o6 or O6/o6) were also analysed and one of them showed recombination between the two loci. Recombination frequency between O6 and b-32 loci, calculated according to the maximum likelihood method, was equal to 4.01.6%. This finding indicates that the O6 locus does not encode the b-32 protein.

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