RESUMO
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.
RESUMO
The 13S globulin zero-repeat subunit is resistant to trypsin and may have higher allergenicity than the 1-6 tandem repeat subunits in common buckwheat (Fagopyrum esculentum Moench). To explore alleles useful for lowering allergenicity, amplicon deep sequencing targeting the zero-repeat subunit gene was conducted in bulked genomic DNA from eight cultivars and landraces. The analysis identified a unique allele encoding a zero-repeat subunit with 10 amino acid insertion (10aa) at a position equivalent to the tandem repeat insertion. Prediction of its 3-D structure suggested that 10aa changes the ß-hairpin structure in the non-10aa (native) subunit to a random coil, which is also found in 1- and 3- repeat subunits. Homozygotes of the 10aa allele were developed and showed that the 10aa subunit was more digestible than the native subunit. However, the 10aa subunit was still less digestible than the 1-6 repeat subunits, suggesting needs to explore unfunctional alleles.
