Isolation of a new member of the soybean Kunitz-type proteinase inhibitors.

Abstract

Proteinase inhibitors are present in almost a11 organisms (Ryan, 1981). Most of these proteins are specific in their interaction with proteinases, inhibiting the proteolytic activity. In plants, this protein family is highly abundant in seeds and tubers of many species. Since high levels of proteinase inhibitor in agricultura1 crops may result in alterations of the digestive enzymes in human and animals, research has been targeted toward modification of the proteinase inhibitor levels in those plant species (Ryan, 1981). In soybean (Glycine max), among others, two important families of proteinase inhibitors have been characterized: the Kunitz inhibitor, which shows specificity for trypsin (Ryan, 1981; Kim et al., 1985), and the Bowman-Risk, which inhibits trypsin, chymotrypsin, and elastase (Ryan, 1981, 1988; Tan-Wilson, 1988). These proteins serve an important function in storage protein metabolism by regulating the level of proteinase activity during seed development (Ryan, 1988). It has been shown that they also play an important role against pathogen attack by acting as endogenous insecticides (Hilder et al., 1986). During screening of a Agtll cDNA library, we isolated a cDNA clone with an insert size of 802 bp. This insert contains a unique open reading frame of 624 bp, which encoded for a 208-amino acid polypeptide (Table I). Sequence comparison with other proteins contained in the data base shows a high level of homology to the Kunitz trypsin inhibitor family. Southern blot analysis of the soybean Kunitz trypsin inhibitor indicates that this protein family contains at least 10 members, some of which are organized in tandem pairs (Jofuku and Goldberg, 1989). At present, four of these members have been cloned and characterized (Jofuku and Goldberg, 1989; Song et al., 1993). The KTi3 and KTi-b genes encode for the major Kunitz trypsin inhibitor proteins, Tia and Tib, respectively (Kim et al., 1985). Studies of the active site indicated that two amino acids, (Pl) and Ile64 (Pl’), are essential for activity. Thus, trypsin inhibitor activity is detected only Table 1. Characteristics of a cDNA coding for a Kun ih trypsin inhibitor orotein. KTi-S