Structure and diversity of 13S globulin zero-repeat subunit, the trypsin-resistant storage protein of common buckwheat (Fagopyrum esculentum M.) seeds.

The zero-repeat subunit of 13S globulin, which lacks tandem repeat inserts, is trypsin-resistant and suggested to show higher allergenicity than the other subunits in common buckwheat (Fagopyrum esculentum Moench). To evaluate allelic variations and find novel alleles, the diversity of the zero-repeat genes was examined for two Japanese elite cultivars and 15 Pakistani landraces. The results demonstrated that two new alleles GlbNA1 and GlbNC1, plus three additional new alleles GlbNA2, GlbNA3, and GlbND, were identified besides the already-known GlbNA, GlbNB, and GlbNC alleles. In the Pakistani landraces, GlbNA was the most dominant allele (0.60-0.88 of allele frequency) in all except one landrace, where GlbNB was the most dominant allele (0.50 of allele frequency). Similar to GlbNC, the alleles GlbNA2 and GlbNA3 had extra ~200 bp MITE-like sequences around the stop codon. Secondary structure predictions of a sense strand demonstrated that the extra ~200 bp sequences of GlbNC, GlbNA2, and GlbNA3 can form rigid hairpin structures with free energies of -78.95, -67.06, and -29.90 kcal/mol, respectively. These structures may affect proper transcription and/or translation. In the GlbNC homozygous line, no transcript of a zero-repeat gene was detected, suggesting the material would be useful for developing hypoallergenic buckwheat.

Elimination of zero-repeat subunit in allergenic seed protein 13S globulin using the novel allele GlbNB2 in common buckwheat (Fagopyrum esculentum Moench).

Common buckwheat (Fagopyrum esculentum Moench) seeds contain 13S globulin, the zero-repeat subunit of which is trypsin-resistant and allergenic. Here, its two novel alleles were analyzed for development of hypoallergenic plants. The GlbNC allele has a Miniature Inverted-repeat Transposable Element (MITE)-like insertion in the 4th exon. However, most of the insertion was spliced-out, resulting in accumulation of zero-repeat subunit in GlbNC homozygotes. Meanwhile, the GlbNB2 has a 164-bp insertion in the 3rd exon, resulting in no accumulation of zero-repeat subunit in GlbNB2 homozygotes (NB2_homo). Both the insertion sequences were predicted to form a hairpin-like structure, and that of GlbNB2 was more rigid than that of GlbNC. Trypsin digestion in NB2_homo showed that the α polypeptide of Met-rich subunit is also hard to digest, that is a next target to eliminate for hypoallergenic buckwheat development.