full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (2) 1149-1156 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1151Fig. 1 Strategy of gene targeting through homologous recombination in brinjal (S. melongena L.). A. Vectorconstruct (pF3GT-F) for targeted integration of nptII and cry1F gene at F3G locus by homologous recombination,GUS expression cassette lies out of homologous recombination cassette (region between two X), B.showing homologous recombination between T-DNA and F3G locus in brinjal genome, homologous recombinationevents will not have GUS gene integration in plant genome, C. F3G locus after homologous recombinationmediated integration of nptII and cry1F genes.(pH 8) and X-Gluc (52.0 gm/ml) in 24 wellplates and incubated overnight at 37°C,followed by incubation in 70% ethanol for fourhours to remove chlorophyll.Molecular Analysis : Plant genomic DNA wasisolated from T-0 plants (28), and screened forGUS, nptII and cry1F gene using a modified PCRprotocol for testing Agrobacterium contaminatedtransgenic plants (29). Following gene specificprimer sequences were used - nptII forwardprimer (5’-CAA TCG GCT CTG ATG CCG-3’)reverse primer (5’-AGG CGA TAG AAG GCGATG CGC-3’), GUS forward primer (5’-GACTGT AAC CAC GCG TCT GTT GAC-3’)reverse primer (5’-AGC CAT GCA CAC TGATAC TCT TCAC-3’), cry1F forward primer (5’-GGA GTG GGA GTG GCG TTT GGC CTG-3’)reverse primer (5’-CCA GTT TGT TGG AAGGCA ACT CCC-3’). These primer sets weredesigned for the amplification of 0.74 kb, 1.2 kband 1.0 kb fragments of nptII, GUS and cry1Fgenes, respectively. The PCR conditions wereas follows: Initial denaturation at 94°C for 4 min;followed by 33 cycles of 94°C (1 min), 55°C (1min), and 72°C (1 min) and final extension at72°C for 5 min.Results and DiscussionTarget site selection : Large variations inexpression and stability among different randomintegration events of transgenic plants developedby T-DNA integration are caused by genomiccontext in which the transgenes have integrated(6). Genes integrated in heterochromatin generallyget silenced due to methylation of promoter regionor packing of DNA into chromatin (6), makingthe promoter inaccessible to RNA polymerasesand transcription factors. Targeted geneintegration at a predetermined location in the plantgenome by homologous recombination providesa mechanism to develop transgenics withpredictable expression. However, expression ofthe transgene depends on the selection of targetingsite used for HR based heterologous geneBt gene targeting in Brinjal