Characterization of a soybean [ Glycine max (L.) Merr.] mutant with reduced levels of Kunitz trypsin inhibitor

Abstract

Kunitz trypsin inhibitor, an abundant soybean [ Glycine max (L.) Merr.] seed protein, has a molecular mass of 21 500 Da and is specific for serine proteases. A soybean mutant (P.I. 196168) was characterized to determine the molecular basis for reduced Kunitz trypsin inhibitor levels during seed development. Western blot analysis revealed that P.I. 196168, in comparison to Amsoy 71, accumulated low amounts of Kunitz trypsin inhibitor protein. Non-denaturing polyacrylamide enzyme activity gels indicated that Amsoy 71 seeds contained at least five distinct zones of trypsin inhibitor activity. However, P.I. 196168 contained only four zones of enzyme inhibition. The coding region of the most abundant trypsin inhibitor gene (KTi3) was isolated from Amsoy 71 and P.I. 196168 by PCR. DNA sequence comparisons of the Kunitz trypsin inhibitor coding regions revealed two deletions and one G to T transversion have occurred. These mutations introduced four stop codons in the reading frame, resulting in a truncated protein. Northern blot analysis revealed that P.I. 196168 accumulated drastically lower amounts of KTi3 mRNA when compared with Amsoy 71.