Class I chitinases in cotton (Gossypium hirsutum): characterization, expression and purification

Chitinases help defend plants from pathogens. We have previously reported t he isolation of a cDNA clone and a genomic clone of Class I chitinase genes from cotton (Gossypium hirsutum). Here we report the results of further in vestigations of Class I chitinase genes in cotton. These include the charac terization of an additional cDNA clone, an estimate of the number of copies in the cotton genome, and experimental evidence for ethylene-induced expre ssion. The Class I chitinase genes have very similar nucleotide sequences ( over 98% identity). Based on Southern analysis, we estimate that there are approximately four copies of the cotton chitinase per haploid genome. Analy sis of total RNA extracted from ethylene-treated plantlets showed that chit inase transcript levels increase after 12 h of treatment. A similar to 31 k Da protein present in ethylene treated extracts has chitinolytic activity a nd cross-reacts with a rabbit antiserum raised against a cotton chitinase f usion protein. Using chitin affinity chromatography of ethylene treated ext racts, we purified a 31.5 kDa protein band with chitinolytic activity. This protein band has been confirmed to be composed of Class I cotton chitinase by N-terminal peptide sequence analysis. The first 20 amino acids are iden tical to those predicted for the Class I cotton chitinases based on our cDN A and genomic nucleotide data. The purified Class I cotton chitinase prepar ation was analyzed by two dimensional electrophoresis. Three different isoe lectric isomers were present in stained 2-D gels, and all three proteins cr oss-reacted with the cotton chitinase fusion protein antiserum. The predomi nant constituent of the affinity purified 31.5 kDa preparation has an isoel ectric point of similar to7.0 and is glycosylated. There are additional iso electric isomers with a pls of 6.2 and 5.8 that are not glycosylated. (C) 2 001 Elsevier Science Ireland Ltd. All rights reserved.