full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (1) 1064-1072 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)Survival Selections Reveal Altered PharmacologicalPhenotypes in Nicotiana tabacum var. SR1 ActivationTagged Mutants1064S. K. Gunjan 1 *, T. Rogers 1,2 , J. Czarnecki 1 , J. Lutz 1 and J. Littleton 1,21Kentucky Tobacco Research and Development Center, 2 Department of Pharmaceutical Sciences, University ofKentucky, Lexington, Kentucky 40546-0236, USA*For correspondence - samir.gunjan@uky.eduAbstractThis study demonstrates the utility ofsurvival-selection strategies for identifyingnovel secondary metabolic profiles andaccompanying pharmacological activity inactivation-tagged mutants generated fromNicotiana tabacum var. SR1. Leaf discs wereinfected by Agrobacterium tumefaciens strain3850 harboring an activation tagging vectorpPCVICEn4HPT in which four copies ofenhancer sequences are located at the rightborder of T-DNA. After Agrobacteriuminfection and co-cultivation, the transformedleaf discs were grown on shoot regenerationmedia containing either ethanol (200 mM) or4-methyltryptophan (4-MT, 50 mM). Thebiochemical analysis of ethanol resistantmutants showed the presence of high level ofantioxidants in plants. Similarly, the mutantswere selected on 4-MT containing mediarevealed 3-5 fold higher nicotine contentcompared to wild-type control plants. Thesestudies demonstrate the utility of survivalselection for identifying individual mutantswith novel pharmacological phenotypes froma large activation-tagged mutant population.Similar selection strategies may be applied toplants of pharmaceutical importance to identifynovel phytochemical drug leads, or possiblyimprove yields of commercially importantsecondary metabolites.Key words: 4-methyltryptophan, activationtagging mutagenesis, antioxidants, ethanol,Nicotiana tabaccum.IntroductionThe elucidation of plant genomicinformation offers tremendous potential forexpanding use of plants as drug discovery andmanufacturing resources. Generation of novelplant compounds or increasing concentrationsof existing economically valuable plantsecondary metabolites is one immediateapplication of our increased understanding ofplant genomics. One strategy for applying theadvances in our understanding of plantgenomic is to use mutagenesis to producelarge mutant populations followed bymetabolic profiling to identify novelmetabolites. Walden and colleagues developeda directed way to induce gain-of-functionmutations in plants (1) that uses four copiesof enhancer elements from cauliflower mosaicvirus 35S gene cloned into T-DNA. Thisapproach, known as activation tagging, cancause transcriptional activation of genes nearits point of insertion into a plant genome.When combined with gene rescue techniques,activation tagging can be a powerful tool foridentifying plant mutants and correspondinggenes. The functions of several genes haveSurvival selection of N. tabaccum activation tagged mutants