Abstract
Agricultural crops suffer many
diseases, including fungal and
bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related
protein genes, such as
chitinases, can
lead to reduction in pathogen
growth, thereby increasing tolerance against fungal pathogens. In the present study, the
chitinase I
gene was isolated from the genomic
DNA of
Barley (
Hordeum vulgare L.) cultivar, Haider-93. The isolated
DNA was used as template for the amplification of the ∼935 bp full-length
chitinase I
gene. Based on the sequence of the amplified
gene fragment, class I
barley chitinase shares 93%
amino acid sequence homology with class II
wheat chitinase. Interestingly,
barley class I
chitinase and class II
chitinase do not share
sequence homology. Furthermore, the amplified fragment was expressed in
Escherichia coli Rosetta
strain under the control of T7 promoter in
pET 30a vector. Recombinant
chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of
IPTG. Expressed
recombinant protein of 35 kDa was purified to homogeneity with
affinity chromatography. Following purification, a
Western blot assay for recombinant
chitinase protein measuring 35 kDa was developed with His-tag specific
antibodies. The purified recombinant
chitinase protein was demonstrated to inhibit significantly the important phytopathogenic
fungi Alternaria solani,
Fusarium spp,
Rhizoctonia solani and
Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.