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

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

Molecular identification and cloning of organophosphate degradation gene in some bacterial isolates

Seventeen local bacterial isolates which can hydrolyze a wide range of organophosphate [OP] insecticides were purified. They were reduced to ten different isolates based on their RAPD pattern and protein profile and termed as ASM-1 through ASM-10. Chemical assay and bioassay revealed that the isolate ASM-5 was the best isolate in chlorpyrifos degradation. The morphological and molecular identification characterized ASM-5 as Agrobacterium tumefaciens. A gene encoding a protein involved in OP hydrolysis was cloned and sequenced from A. tumefaciens ASM-5. This gene [named opdA] had sequence similarity about 98.7% with the A. tumefaciens opd gene. The coding sequence of the gene was sub cloned down stream an inducible expression promoter to evaluate the gene-enzyme system responsible for its OP-hydrolyzing activity. The biological activity of OPDA protein became more efficient compared to OPDA native protein