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Current Trends in Biotechnology and PharmacyVol. 5 (2) 1149-1156 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1149Targeted Integration of Bacillus thuringiensis δ-Endotoxincry1Fa1 in Brinjal (Solanum melongena L.)Dipty Shrivastava, 1 Monika Dalal, 1$ Vikrant Nain, 1# P. C. Sharma, 2 P. Ananda Kumar* 11National Research Centre on Plant Biotechnology, IARI, New Delhi -110012, India.2School of Biotechnology, GGS Indraprastha University, Sector -16C Dwarka Delhi, India.Present addresses - #School of Biotechnology, Gautam Buddha University, Greater Noida-201308, India$National Research Centre for Sorghum, Rajendra Nagar, Hyderabad-500 030, India*For Correspondence - kumarpa@nrcpb.orgAbstractGene integration in plants by homologousrecombination eliminates variation in transgeneexpression and gives a predictable plantphenotype. Present study demonstrateshomologous recombination-mediated integrationof Bacillus thuringenisis cry1Fa1 gene atFlavonoid-3-glucosyltransferase (anthocyaninbiosynthesis pathway) locus in Brinjal (Solanummelongena L.), for introduction of resistanceagainst its major insect pest ‘Brinjal Fruit andShoot Borer’ (BFSB) (Leucinodes orbonalis).The transgenic lines showed complete protectionagainst BFSB. These results provide a novelperspective on development of geneticallymodified crops with stable and predictabletransgene expression, and the possibility of theutilization of such promising technology in futurecrop improvement programmes.Key words: Anthocyanin, Bt transgenic, Genetargeting, Homologous recombination, cry1F,Leucinodes orbonalis.IntroductionTechnological developments in the field ofplant molecular biology and biotechnology haverevolutionized crop improvement (1). Availablegene pool of all the living organisms makes itpossible to introduce beneficial genes from anyspecies to the desired crop varieties (2). Standardplant genetic transformation techniques areAgrobacterium-mediated transformation andparticle bombardment (3). In both of theseapproaches random transgene integration byillegitimate recombination in the genome resultsin considerable variation in transgene expressionacross different integration events and variationin transgene copy number (4). In addition, randomT-DNA integration induces undesirable mutationsin the host genome with unpredictable phenotypes(5). Gene targeting is a potential technology thatcan be used for making transgenic plants withpredictable transgene expression and changes inhost plant genome (6). It is based on homologousrecombination, which allows the integration ofDNA at predetermined positions, and thereforefacilitates precision genome manipulation (7, 8).Gene targeting has been efficiently appliedto modify the genomes of bacteria (9), fungi (10)and eukaryotes (11), especially mammals wherethe targeting frequencies of 10 -2 have beenreported (12, 13). In plants a gene targetingfrequency of only 10 -6 to 10 -3 have been reported.(14, 15,16,). Different strategies that includeextending the homology length in targeting vectors(17) and using strong positive-negative selection toenrich the targeted events had been tested (18,19). Shukla et al. (21) and Townsend et al. (20)Bt gene targeting in Brinjal
Current Trends in Biotechnology and PharmacyVol. 5 (2) 1149-1156 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1150described modification of maize gene IPKI andtobacco gene SuRB gene respectively (20, 21).Shukla et al. (21) introduced an herbicideresistance gene into the IPKI locus and foundthat 3-20 percent of the resistant samples hadIPKI targeted integration. Townsend et al. (20)used homologous recombination to target smallchange in the SuRB gene that resulted in achievinggene-targeting rates of four percent.Since the first report on transgene integrationby HR in plants (22), with the exception of a recentreport by Shukla et al. (21), all attempts werelimited to the demonstration of either integrationof an exogenous gene at a predefined locus withincreased frequency or screening rare HR eventsfrom random integration events (23). The presentstudy was carried out carried out to harness thebenefit of gene targeting by developing insectresistant Bt transgenic brinjal (Solanummelongena).Brinjal (Solanum melongena L), animportant vegetable crop is severely infested byBrinjal Fruit and Shoot Borer (BFSB)(Leucinodes orbonalis) (Lepidoptera: Pyralidae)(24). The B. thuringenisis cry1F gene has beenfound to be very effective against BFSB (Kumaret al. unpublished) and hence was selected forintroduction in brinjal. For screening out therandom T-DNA integration events at early stagesof transgenic plant development, GUS reportergene was used as a negative screening marker.Two targeted gene integration events wererecovered out of 954 random gene integrationevents. The present study successfullydemonstrated genetic modification of a crop plantfor insect resistance by gene targeting.Materials and MethodsVector construction for homologousrecombination :Brinjal cv. Pusa Purple Long wasused as the host plant for carrying out the genetargeting experiments. Flavonoid-3-glucosyltransferase (F3G) gene fromanthocyanin synthesis pathway, which catalyzesthe conversion of flavonoids into glycosides, wasselected as a target locus. The target gene F3Gwas PCR amplified from brinjal genomic DNAusing gene specific primers based on the publishedsequence (GenBank accession no. E12713). ThePCR amplicon (1.3 kb) was cloned intopGEMTeasy vector (Promega) and referred aspF3G-TA. To develop HR mediated geneintegration vector, F3G gene from pF3G-TAvector was sub-cloned in pCAMBIA1304 usingXbaI (vector)-XhoI (insert) blunt at 5’end andSalI site at 3’end. pBI121∆GUS vector wasgenerated by deleting GUS gene from pBI121vector. In the next step, nptII expression cassetteand CaMV35S promoter with NOS terminatorfrom pBI121∆GUS were excised out with EcoRIand PmeI, end-filled and cloned at end-filledBamHI site (640 bp) of F3G and the constructwas refered as pF3GT (Fig. 1). The syntheticcodon modified cry1F gene was cloned in XbaI-BamHI sites present between CaMV35Spromoter and NOS terminatior, downstream tonptII expression casette. This final constructnamed as pF3GT-F, was used as the gene targetingconstruct.Genetic transformation of brinjal : Binaryvector pF3GT-F was mobilized in Agrobacteriumtumefaciens strain EHA-105 by freeze-thawmethod (25). Brinjal was genetically transformedby using cotyledon leaf explants (26). The rootedkanamycin resistant plants were transplanted topots, acclimatized and maintained in a glasshouse.Histochemical GUS assay : Third leaf fromputative transformants was analyzed for GUSactivity (27), for screening the T-DNAintegration events. Leaf tissues were immersedin 0.5 ml GUS assay buffer (0.5M EDTA,Triton X-100, 0.1M Sodium phosphate bufferDipty Shrivastava et al
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(December 7-9, 2011)Venue: Karunya
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