full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
- No tags were found...
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (2) 1149-1156 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1151Fig. 1 Strategy <strong>of</strong> gene targeting through homologous recombination in brinjal (S. melongena L.). A. Vectorconstruct (pF3GT-F) for targeted integration <strong>of</strong> nptII <strong>and</strong> cry1F gene at F3G locus by homologous recombination,GUS expression cassette lies out <strong>of</strong> homologous recombination cassette (region between two X), B.showing homologous recombination between T-DNA <strong>and</strong> F3G locus in brinjal genome, homologous recombinationevents will not have GUS gene integration in plant genome, C. F3G locus after homologous recombinationmediated integration <strong>of</strong> nptII <strong>and</strong> cry1F genes.(pH 8) <strong>and</strong> X-Gluc (52.0 gm/ml) in 24 wellplates <strong>and</strong> 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), <strong>and</strong> screened forGUS, nptII <strong>and</strong> 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 <strong>of</strong> 0.74 kb, 1.2 kb<strong>and</strong> 1.0 kb fragments <strong>of</strong> nptII, GUS <strong>and</strong> cry1Fgenes, respectively. The PCR conditions wereas follows: Initial denaturation at 94°C for 4 min;followed by 33 cycles <strong>of</strong> 94°C (1 min), 55°C (1min), <strong>and</strong> 72°C (1 min) <strong>and</strong> final extension at72°C for 5 min.Results <strong>and</strong> DiscussionTarget site selection : Large variations inexpression <strong>and</strong> stability among different r<strong>and</strong>omintegration events <strong>of</strong> 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 <strong>of</strong> promoter regionor packing <strong>of</strong> DNA into chromatin (6), makingthe promoter inaccessible to RNA polymerases<strong>and</strong> transcription factors. Targeted geneintegration at a predetermined location in the plantgenome by homologous recombination providesa mechanism to develop transgenics withpredictable expression. However, expression <strong>of</strong>the transgene depends on the selection <strong>of</strong> targetingsite used for HR based heterologous geneBt gene targeting in Brinjal