A WOUND-INDUCIBLE GENE FROM SALIX-VIMINALIS CODING FOR A TRYPSIN-INHIBITOR

A gene designated swin1.1 has been isolated by screening a Salix vimin alis genomic library with a heterologous probe, win3 from Populus. The region sequenced included the entire coding sequence for a protein wi th 199 amino acids plus the promoter and terminator. At the 5' end of the coding region is a sequence that encodes a hydrophobic region of 2 5-30 amino acids, that could form a signal peptide. A putative TATAA b ox and polyadenylator sequence were identified. Introns were absent. T he gene product showed similarities with serine protease inhibitors fr om the Kunitz family and especially with win3 from wounded leaves of P opulus. Southern blot analysis indicated that swin1.1 is a member of a clustered gene family, swin1. An oligonucleotide corresponding to the putative hypervariable region towards the carboxyl end when used as a probe in Southern hybridization showed high specificity for swin1.1. Expression of the swin1.1 gene was enhanced in wounded Leaves. The swi n1.1 coding region without the signal sequence was highly expressed in Escherichia coli and the protein showed inhibitory activity against t rypsin but at most slight activity against the other proteases tested. A systemically induced protein, SVTI, with inhibitor activity against trypsin, was isolated from Salix leaves by affinity chromatography on a column of trypsin-Sepharose 4B and N-terminal sequenced. It corresp onded with the translated swin1.1 gene at 16 of the 19 amino acid site s, suggesting that SVTI is encoded by another member of the swin1 gene family.