Thesis (Ph.D.) - Cairo University - Faculty of Agriculture - Department of Agricultural Biochemistry

This study describs cloning of plant defensin (PDF) gene isolated from seedlings of kidney bean (Phaseolus vulgaris L.) cultivar polesta, which designated as pvPDF. The resistant antifungal gene (pvPDF) was isolated directly from total DNA of kidney bean leaf tissue using polymerase Chain Reaction technique (PCR). The corresponding full length gene, named pvPDF was cloned, sequenced and characterized further, confirmed by restriction endonucleases Xba1 and BamH1 analysis. Its nucleotide sequence consists of 486 bp. The pvPDF sequence analysis showed a high significant homology to other known plant defensin gene sequences that presented in the database using the BLAST program. The pvPDF sequence has been deposited in the Gen Bank database with accession number kj939334. Furthermore, the characterized pvPDF DNA cloned in strata clone pSC-A vector for sequencing using Escherichia coli DH5 alpha competent cells. The presence of intron (non coding regions) in genomic pvPDF was deleted with SOEing PCR techniques. Then the pvPDF DNA sequence was fused to Ý- glucuronidase (GUS) using pBI121 binary vector under control of the CaMV 35S promoter and the NOS terminator region. This whole cassette was used for tobacco plant cells transformation via agrobacterium tumefaciens (LB4404) for pvPDF function validation. Analysis of transgenic pvPDF-GUS tobacco plants indicated that GUS activity was observed with gene constructs with the strongest being in leaf. GUS activity was the highest with pvPDF gene. To verify the expression of pvPDF, it was constructed into pET29a plasmid under the control of T7 promoter then transformed into E. Coli BL21 bacteria. The analysis of resulted protein indicated that the pvPDF gene was expressed at 8 KDa

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