Entity evidence for differentiation between Tia and Tib types of soybean Kunitz trypsin inhibitor: detection of a novel transitional variant type between Tia and Tib in wild soybean (Glycine soja Sieb. & Zucc.)

Soybean Kunit trypsin inhibitor (SKTI) has several polymorphic types. Of these SKTI, there are large differences of nine amino acid substitutions between Tia and Tib. So far no transitional type between them has been found. A novel transitional intermediate variant between Tia and Tib was detected in 11 lines from 720 Japanese wild soybeans (Glycine soja Sieb. & Zucc.). This variant showed identical electrophoretic mobility to Tib in the Davis system polyacrylamide gel electrophoresis (PAGE), but higher electric points than other SKTI proteins (Tia, Tib, Tic) in isoelectric focusing PAGE. The genetic analysis of SKTI in F₂ seeds from a cross between the novel variant type and Tib showed that this variant type is inherited as codominant alleles in a multiple allelic system at an SKTI locus. This variant also showed inhibitory activity to trypsin. We propose the genetic symbol Ti b ⁱ⁵ for this novel variant. The sequence analysis of Tib ⁱ⁵ revealed that six nucleotides were different between Tib ⁱ⁵ and Tia, and the nucleotides of these mutated positions were identical to Tib. This causes substitution of five amino acids at the residue position 62 (Tyr→Phe), 74 (Ser→Arg), 114 (Met→Val), 120 (Leu→Ile) and 137 (Pro→Thr). These substitutive amino acids are completely in accord with the amino acids of Tib, showing that Tib ⁱ⁵ is an intermediate between Tia and Tib types. Tib ⁱ⁵ type is widely distributed throughout seven separate areas from northeast to southwest Japan with a 1.5% frequency of total materials examined. This indicated that Tib ⁱ⁵ type did not originate from a recent mutation event, but had spread in wild soybean from ancient times.