Arabidopsis motif scanner

Above results confirm that our newly constructed ‘Gene-deletor’ vector driven by LFY promoter can achieve certain exogenous gene elimination effect in Arabidopsis thaliana.Fig. 2Analysis of eliminating effect of ‘Gene-deletor’ vector pBIN19-LFY-FLP in Arabidopsis thaliana. a Transgenic Arabidopsis thaliana homozygous T3 root GUS staining. b GUS staining in the stem of transgenic Arabidopsis thaliana homozygous T3. c GUS staining in leaves of transgenic Arabidopsis thaliana homozygous T3. d GUS staining in fruit pods of transgenic Arabidopsis thaliana homozygous T3. e GUS staining of the fruits of transgenic Arabidopsis thaliana homozygous T3. f The calculated elimination efficiency of exogenous genes in the fruits of transgenic Arabidopsis thaliana homozygous T3. The above experiments were repeated several times. Representative experiments were photographed, and the eliminating efficiency of exogenous genes was calculatedFull size imageGeneration of transgenic banana plants and their molecular analysisIn order to further verify the feasibility of using ‘Gene-deletor’ vector in bananas, ECSs of banana were genetically transformed using A. tumefaciens harboring ‘Gene-deletor’ vector (pCAMBIA-LFY-polseed-FLP) (Fig. 1e). Three subcultures in fresh medium were performed once in 10 days (Fig. 3a). Three months from transformation and selection, whitish embryos emerged on embryo development medium supplemented with 100 mg/L Kanamycin (Fig. 3b). These embryos were later transferred onto germination medium to aid the emergence of first plantlets (Fig. 3c), after which the tiny buds were transferred to multiplication medium. Clusters of small plantlets and white buds were observed after 1 month of culture (Fig. 3d). Single plantlets were isolated and rooted on rooting medium supplemented

LFY promoter ligated to the large fragment after enzyme digestion, and then undergoing BamHI and SalI double enzyme digestion to verify whether the LFY promoter ligated with the vector successfully. d PCR positive identification after recombinant vector transformation of Agrobacterium tumefaciens. e Schematic diagram of the newly constructed ‘Gene-deletor’ vector pCAMBIA-LFY-polseed-FLPFull size imageVerification of the elimination effect of ‘Gene-deletor’ vector in Arabidopsis thalianaIn order to verify the elimination effect of ‘Gene-deletor’ vector exogenous gene driven by the newly constructed LFY promoter, we first conducted genetic transformation verification in A. thaliana. GUS staining was performed on the roots, stems, leaves, fruit clips and fruit tissues of transgenic Arabidopsis thaliana, and GUS activity was observed in the roots, stems, leaves and fruit clips (Fig. 2a–d), indicating that the newly constructed pCAMBIA-LFY-polseed-FLP vector could not achieve the effect of exogenous gene elimination in the roots, stems, leaves and anthers of A. thaliana. Keeping in mind that the LFY promoter is a key promoter affecting flowering and development of Arabidopsis, we hypothesize that the newly constructed ‘Gene-deletor’ vector might perform a certain eliminating function in late flowering bud differentiation phase (fruits) of plants. To test the above hypothesis, we further stained the transgenic Arabidopsis fruits with GUS, and found that of the 200 Arabidopsis fruits, only 23 showed positive GUS staining and 177 showed negative GUS staining (Fig. 2e). The results indicate that the exogenous genes in the non-stained Arabidopsis thaliana fruits were successfully eliminated, with an elimination effect of 88.5% (Fig. 2f). The

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AbstractBanana (Musa spp.) is an important tropical crop. Banana industry is under biotic and abiotic stresses such as Fusarium wilt, typhoon, cold stress. Genetic engineering offers a powerful strategy to create germplasm of banana with enhanced resistance. The safety of genetically modified organisms has become a bottleneck restricting the popularization and application of genetically modified technology. In this study, a candidate promoter, LEAFY (LFY) for expression and flower initiation in Arabidopsis, was cloned and constructed to ‘Gene-deletor’ vector. Histochemical β-glucuronidase (GUS) staining results showed that the ‘Gene-deletor’ vector driven by LFY promoter could lead to 88.5% excision efficiency from Arabidopsis seeds based on more than 200 T3 progeny examined per event. GUS staining was found to be partially negative in transgenic bananas, however, polymerase chain reaction could still detect the presence of large fragments of the vector. These results suggest that although LFY promoter could not completely drive the ‘Gene-deletor’ vector to achieve the effect of complete elimination of exogenous gene in bananas, its efficiency of eliminating exogenous gene laid a theoretical foundation for cloning banana fruit-specific promoters, that is, ‘non-transgenic’ GM bananas.Key messageThese results suggest that LFY promoter could not completely drive the ‘Gene-deletor’ vector to achieve the effect of complete elimination of exogenous gene in bananas. Nevertheless, a certain effect of exogenous gene elimination laid a theoretical foundation for the next step of screening banana fruit-specific promoters, removing all exogenous genes from banana fruits, and solving the food safety problem of genetically modified bananas. Similar content being viewed