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
Subject(s)
Chitinases , Antifungal Agents , Plant LeavesABSTRACT
Barley yellow dwarf viruses [BYDVs] are members of the luteovirus group transmitted only by aphids. The five serotypes [PAV, RMV, RPV, MAV and SGV] were reported. In Egypt, BYDV is common with PAV serotype being dominant. In the current report, total RNA was purified from infected wheat leaf plants and Aphids. RT-PCR technique was used to amplify and identify the coat protein gene sequence of BYDV- PAV and RMV serotypes in wheat using specific primers designed by ABI primer express software. Expected PCR products were sequenced and aligned together with related gene bank sequences and revealed the high similarity up to 93%. RT- Real Time PCR technique was used to detect and quantify BYDV. The results indicate that the infection ratio of Giza 164 samples are higher than the infection ratio of Sids 7 based on Ct value, and virus concentration in aphids are higher than in wheat for both serotypes. In addition, the sensitivity of RT- Real Time PCR is 3 to 5 fold higher than conventional PCR for detecting virus infection