الفهرس 
Biochemical studies on isolation and characterization of nematode resistant genes to produce transgenic plants by molecular biology techniques
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Abstract
The present study was conducted during the period between 2010 and 2015 at Department of Plant Biotechnology and Center of Excellence for Advanced Sciences (CEAS),NRC, Dokki, Cairo, Egypt,. To isolate Nematode resistance gene such as Cry1 Aa and Cry1 Ab genes from local Egyption isolates of Bacillus thuringiensis used PCR amplification and another candidate root-knot nematode resistance gene (standard genes) which were isolated from stander isolate of Bacillus thuringiensis as a control in this study. These genes were cloned then confirmed by restriction analysis and sequencing. Results showed that the predicted open reading frame (ORF) had 99% identity to the published CryI Aa and CryI Ab nematode resistance gene. To verify the expression Cry1 Aa and Cry1 Ab, these genes were constructed into pET 29a plasmid under the control of a T7 promoter then transformed into E. coli BL21 bacteria. Results of protein analysis indicated that the Cry1 Aa and Cry1 Ab genes were expressed as 65KDa.Regeneration system has been established for strawberry cvs Diamante on MS medium supplemented with different combinations of growth regulators such as NAA, IAA, TDZ.The obtained results indicated that the best combination of growth regulators for callus induction and regeneration was NAA 0.3/TDZ 4.0 mg/L-1.To verify the function of Cry1 Aa and Cry1 Ab genes, pBI121 binary vector fused to Ý-glucuronidase (gus) containing the genomic DNA spanning the toxic coding region of Cry1 Aa and Cry1 Ab were constructed then transferred into root- knot nematode susceptible strawberry cvs Diamante using Agrobacterium tumefisiense EH105. Putative transformantes regenerated from explants on regeneration medium with antibiotics were screened using gus assay and confirmed by PCR. Gus expression was observed in transformed strawberry shoots but never in non transformed (control). Gus positive explants reacted positively to PCR and the obtained results indicated that the nematode resistance genes of interest CryI Aa and CryI Ab were introduced in strawberry genome