Identification and Characterization of a Heat-stress Induced Gene in Cabbage Encodes a Kunitz Type Protease Inhibitor

Abstract

We have identified a heat-stress induced gene (BocHS 1) by differential screening of a cDNA library constructed from Brassica oleracea var. capitata (cabbage) exposed to high temperature at 35°C. Sequence analysis of BocHS 1 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 total RNA indicates that a single transcript of approximately 1.9 kb was identified in heatshocked plants. No transcript was detected in plants grown at low temperature (20°C). The level of transcript hybridizing to BocHS 1 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°C from 4 to 72 h. The level of gene expression was further monitored at a different range of temperature from 15 to 45°C and the expression of this gene was noticed from 30, 35 and 40°C. This gene is also induced by drought but not by exogenous application of ABA or salt-stress. Transferring plants from 35°C to low temperature (20°C) results in the loss of detectable transcript hybridizing to BocHS 1. Hybridization of the heat-stress induced gene with genomic DNA revealed three copies and we suggest that BocHS 1 is encoded by a small gene family in Brassica. The protein encoded by BocHS 1 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.