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

摘要

Chitinases help defend plants from pathogens. We have previously reported the 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 investigations of Class I chitinase genes in cotton. These include the characterization of an additional cDNA clone, an estimate of the number of copies in the cotton genome, and experimental evidence for ethylene-induced expression. 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. Analysis of total RNA extracted from ethylene-treated plantlets showed that chitinase transcript levels increase after 12 h of treatment. A similar to 31 kDa protein present in ethylene treated extracts has chitinolytic activity and cross-reacts with a rabbit antiserum raised against a cotton chitinase fusion protein. Using chitin affinity chromatography of ethylene treated extracts, 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 identical to those predicted for the Class I cotton chitinases based on our cDNA and genomic nucleotide data. The purified Class I cotton chitinase preparation was analyzed by two dimensional electrophoresis. Three different isoelectric isomers were present in stained 2-D gels, and all three proteins cross-reacted with the cotton chitinase fusion protein antiserum. The predominant constituent of the affinity purified 31.5 kDa preparation has an isoelectric point of similar to7.0 and is glycosylated. There are additional isoelectric isomers with a pls of 6.2 and 5.8 that are not glycosylated.