A class I chitinase from soybean seed coat

Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fract ionated by isoelectric focusing and SDS-PAGE analysis and components identi fied by peptide microsequencing. An abundant 32 kDa protein possessed an N- terminal cysteine-rich hevein domain present in class I chitinases and in o ther chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase act ivity by an electrophoretic zymogram assay. The corresponding cDNA and geno mic clones for the chitinase protein were isolated and characterized, and t he expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conse rved chitin-binding and catalytic domains interspaced by a proline hinge. A n 11.3 kb EcoR1 genomic fragment bearing the 2.4 kb chitinase gene was full y sequenced. The gene contained two introns and was flanked by A+T-rich tra cts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed d evelopment, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study fur ther illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in th e seed coat.