5183
EMAN ABDEL MOTTALEB MAHMOUD
BIOCHEMICAL STUDIES ON ISOLATION AND CHARACTERIZATION OF ANTIFUNGAL GENE AND TRANSFERRING TO PLANTS
Gerre Isolation, Cloning, Phaseolus t+lgaris. Defensin Gene, pvPDF. gene constmct.
This study describs cloning of plant defensin (PDF) gene isolated from seedlings of kidney bean (Phaseolus ralgaris L.) cultivar polesta, n'hich 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 coresponding full length gene, named pvPDF was cloned, sequenced and characterized further, confinned by restriction endonucleases )Oal and Barrl{l anaiysis. lts nucleotide sequence consists of 486 bp. The pvPDF sequence analysis showed a high significant homology to other knorvn plant defensin gene sequences that presented in the database using the BLAST program. The pvPDF sequence has been deposited in the GenBank database rvith accession number k1939334. Furthermore, the characteized puPDF DNA cloned in strata clone pSC-A vector for sequencing using Escherichia coli DHS alpha competent cells. The presence of intron (non coding regions) in genomic pUPDF was deleted witli SOEing PCR techniques. Then the pvPDF DNA sequence was fused to B- glucuronidase (GUS) using pBI121 binary vector under control of the CaMV 35S promoter and the NOS terminator region. This rvhole cassette was used for tobacco plant cells transformation via Agrobacreriunt tumefacrens (L84404) for ptPDF function validation. Analysis of transgenic pvPDF-GUStobacco plants indicated that GUS activity r,vas 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 tlre control of T7 prorloter then transfoliled into E ColiBL2l bacteria. The anaiysis of resulted protein indicated that the pvPDF gene wasexpressed at 8 KDa.
2016
Ph.d
Cairo
Agriculture