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)1152integration. To develop transgenic brinjal for insectresistance, Bt gene driven by strong CaMV35Spromoter must express in leaf, young stem andfruits at considerably higher level. It is possibleonly when the gene locus selected for targetedintegration of Bt gene is under favorable chromatinstructure or context for transcription in all thetarget tissues.In the present study, F3G gene, a keyenzyme in anthocyanin biosynthesis pathway wasselected as a locus for gene targeting.Anthocyanin pigment is present in leaf vein, youngstem, flower and fruit (Fig. 2A-B). RT-PCRanalysis also confirmed that F3G gene is expressedin leaves, young stems, flowers (petal) and fruitepidermis (Fig. 2C). So, F3G locus can be asuitable genomic context for expression of cry1Fgene, driven by a constitutive promoter. F3G isencoded by an intron-less gene of 1.3 kb lengthand there are 3 copies of F3G in S. melongenagenome (Fig. 2D). Hence, disruption of singlecopy of F3G gene may not have pleiotropic effectson the plant. The active transcription of F3G inall the desired tissues was presumed to provide afavorable genomic context for expression ofcry1F gene and presence of two functional F3Gcopies after loss of one copy of F3G by targetedgene integration may not have any significanteffect on anthocyanin synthesis. This strategyFig. 2 Analysis of F3G expression and gene copy number in brinjal. A-B. anthocyanin pigmentation in leafvein (LV), flower (F) and B. fruits, C. RT PCR for F3G transcript in 1. leaf, 2-3. stem and fruit epidermal tissuesand 4. petal, showing expression of F3G gene in these tissues, D. Southern analysis indicating three copiesof F3G gene in brinjal genome.Dipty Shrivastava et al