Overexpression of rice chitinase gene: evaluation of chitinase ability as a bio-antifungal agent

ABSTRACT

Seven local fungal isolates of Pyricularia grisea were purified from infected rice leaves. The total proteins were extracted and SDS-PAGE was carried out to differentiate between the expression of proteins in infected and healthy plants. SDS-PAGE analysis revealed the accumulation of 35-kDa chitinase after 16, 20, 24 and 48 hr [hours post inoculation]. Rice chitinase gene [1.023 bp] was successfully amplified from the total RNA extracted from infected rice using RT-PCR. The amplified fragment was cloned and overexpressed in E. coli BL21 cells as 6x-His- fusion protein. Recombinant chitinase fusion protein was successfully purified using Ni-NTA affinity column chromatography. Two chitinase activity assays against P. grisea were carried by the filter disc and the dissimilar concentrations plates method. The results indicated that the expressed chitinase protein had an antifungal activity against P. grisea