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

Evaluation of some cucumber inbred lines and their hybrids for cucumber mosaic virus [CMV] resistance

The major objective of our cucumber breeding program involves breeding for CMV disease resistance to reduce losses in quality and yield. Seven half diallel cross hybrids resistant to CMV were developed at the Vegetables Breeding Dept., Hort. Res. Inst., Egypt during March, 2006. Sixteen genetically different pure inbred lines of Cucumis sativus were used to develop the hybrids obtained from different sources and selected for their CMV disease-resistance trait. The inbred lines and one commercial [Beit alpha] cultivar as a susceptible control were examined to CMV resistance using biological, serological and molecular methods. The CMV isolate identified by Plant Path. Res. Inst. Virus and Phytoplasma Res. Dept. [ARC] was used in mechanical inoculation of all cucumber genotypes used during this study. The seeds of the genotypes were incubated and the seedlings were cultivated in foam trays with peat soil and kept under greenhouse conditions. At the cotyledon stage, i.e. before the development of the first true leaf, the seedlings were mechanically inoculated by rubbing with virus inoculum. Disease severity was assessed visually 7- 10 days [on cotyledons] and 14-25 days [on true leaves] after inoculation with CMV. The results revealed that six out of sixteen cucumber inbred lines [Cus 260/1980, 6-5-23-2 Kaha, 1-180-309- 18-105 Dokky, 5-57-22-17 Kaha, Cus 38/1991, and 25-2-1-90 Kaha] were found to be without systemic symptoms of CMV infection and proved to be resistant to CMV when tested by DAS-ELISA and RT-PCR. The promising accessions as sources of resistance have been intercrossed with leading commercial type [Beit-alpha] in half diallel system. In order to determine the genetic polymorphism and discriminate between cucumber inbred lines, RAPD-PCR analyses were conducted on the DNA isolated from each line. Dendrograms representing genetic distances were performed on the studied genotypes using the UPGMA [Unweighted Pair Group Method with Arithmetic Average]. Twenty one cucumber hybrids obtained from the half diallel crossing between the six resistant genotypes and the local commercial cultivar [Beit-alpha] were subjected to CMV artificial inoculation in a separate greenhouse and symptoms were visually monitored for two months. Only seven cucumber hybrids showed high a level of resistance to CMV were screened in the greenhouse and evaluated for CMV resistance. The resistant hybrids obtained did not develop visual symptoms of CMV infection on cotyledons and true leaves. These resistant lines could serve as potential sources of resistance in breeding programs

Molecular cloning, expression and characterization of mouse single-chain fragment variable antibody against citrus tristeza virus

The sequences encoding the mouse heavy-chain [V[H]] and light-chain [V[L]] variable region genes were isolated by PCR and joined into a single-chain Fv [scFv] DNA by using a DNA linker encoding [Gly4Ser][3] peptide. The scFv DNA fragment was cloned into the phagemid pCANTAB5E and expressed in Escherichia coli as a fusion protein with M13 phage p3 polypeptide and E tag. The scFv fusion protein was displayed on the surfaces of recombinant M13 phages in the presence of the helper phage M13K07. High-affinity scFv phage-bodies against citrus tristeza virus [CTV] were enriched through affinity selection on immobilized recombinant CTV coat protein preparations. The selected recombinant phages were used to infect Escherichia coli HB2151 for the production of soluble scFv antibodies. One selected clone in HB2151 secreted a soluble scFv antibody that detected CTV in extracts of infected citrus plants with a sensitivity comparable to that of a commercial monoclonal antibody. The nucleotide sequence of the light-chain and heavy-chain portions were closely related to other published scFv against CTV. The potential of this scFv was demonstrated in routine field testing using an inexpensive tissue print-ELISA