Plant Science 160 (2001) 979–986 Characterization of a soybean [Glycine max (L.) Merr.] mutant with reduced levels of Kunitz trypsin inhibitor Hari B. Krishnan * USDA-ARS, Plant Genetics Research Unit and Department of Agronomy, 108W Curtis Hall Uniersity of Missouri, Columbia, MO 65211, USA Received 2 November 2000; received in revised form 18 December 2000; accepted 3 January 2001 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. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Anti-nutritional protein; Glycine max; Storage protein; Trypsin inhibitor www.elsevier.com/locate/plantsci rence of Ti a decreased as the maturity group num- ber increased. The genetic organization and developmental regulation of KTi expression have been examined in detail [7–10]. It has been estimated that the soybean genome contains at least ten distinct KTi genes, some of which occur in tandem pairs. Three of the KTi genes (KTi1, KTi2, and KTi3) have been cloned and sequenced. Nucleotide sequence analysis revealed that the KTi1, KTi2 and KTi3 genes contain no introns [8]. KTi3 encodes the predominant trypsin inhibitor in soybean seeds. The KTi3 nucleotide sequence has diverged ap- proximately 20% from those of KTi1and KTi2. In addition to soybean seeds, the KTi1and KTi2 mRNAs are expressed in other organs, such as leaves, stem, and roots, but at 1000-fold lower levels than mid-maturation stage embryos. In con- 1. Introduction Two classes of proteinase inhibitors, Kunitz and Bowman – Birk, are found in soybean seeds [1,2]. The most abundant, a Kunitz inhibitor (KTi) of 21 kDa, specifically inhibits trypsin [3,4]. The Bowman – Birk (BBTi) class of proteinase inhibitor comprises several related proteins that are specific for trypsin, chymotrypsin, and elastase [4,5]. Hy- mowitz [6] identified three basic variants of KTi (Ti a , Ti b , and Ti c ) and determined that 89% of USDA soybean collection contained the Ti a vari- ant. In contrast, Ti c was found in only 0.3% of the collection. In addition, he reported that the occur- * Tel.: +1-573-8828151; fax: +1-573-8847850. E-mail address: krishnanh@missouri.edu (H.B. Krishnan). 0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII:S0168-9452(01)00346-6