Identification and characterization of a heat-stress induced gene in cabbage encodes a Kunitz type protease inhibitor

We have identified a heat-stress induced gene (BocHS1) by differential screening of a cDNA library constructed from Brassica oleracea var. capitata (cabbage) exposed to high temperature at 35 degrees C. Sequence analysis of BocHS1 revealed that it encodes a putative protein related to 22 kDa drought and salt-induced proteins. These proteins exhibited high similarity to the Kunitz type of protease inhibitors. Northern blot analysis of coral RNA indicates that a single transcript of approximately 1.3 kb was identified in heat-shocked plants. No transcript was detected in plants grown at low temperature (20 degrees C). The level of transcript hybridizing to BocHS1 increased with 24-h exposure to high temperature. Further, a high level of transcript was also noticed with the other cultivars that had been heat-shocked at 35 degrees C frdm 4 to 72 h. The level of gene expression was further monitored at a different range of temperature from 15 to 45 degrees C and the expression of this gene was noticed from 30, 35 and 40 degrees C. This gene is also induced by drought but not by exogenous application of ABA or salt-stress. Transferring plants from 35 degrees C. to low temperature (20 degrees C) results in the loss of detectable transcript hybridizing to BocHS1. Hybridization of the heat-stress induced gene with genomic DNA revealed three copies and we suggest that BocHS1 is encoded by a small gene family in Brassica. The protein encoded by BocHS1 exhibits a new class of genes that are related to protease inhibitors; also, this is the first example of the Kunitz type of protease inhibitors induced in vegetative tissues during heat-stress.