Download arabidopsis motif scanner

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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

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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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University of Connecticut developed the ‘Gene-deletor’ technology. This technique uses the pollen and seed-specific PAB5 promoters, which automatically eliminate all foreign genes in the pollen and seeds before and after transgenic flowering. This solves the problem of genetic flow of transgenic plants, and solves the concern about the safety of genetically modified food (Li et al. 2007). The efficacy of this technique has been demonstrated in tobacco (Luo et al. 2007).Recently, Chong-Perez et al. (2013) reported that the feasibility of using developmentally controlled promoters to mediate marker excision in banana. Considering that the LEAFY gene is the key gene affecting plant flowering development, in order to complete the removal of exogenous genes in early fruit formation, in this study the LFY promoter of the flower-determination gene LEAFY from A. thaliana genome was cloned (Fig. 1a), and successfully replaced the PAB5 promoter in the exogenous ‘Gene-deletor’ vector pCAMBIA-PAB-polseed-FLP (Fig. 1b). In order to further verify the eliminating effect of the newly constructed exogenous gene vector pCAMBIA-LFY-polseed-FLP in fruits, we first carried out genetic transformation in A. thaliana. Through GUS staining of the roots, stems, leaves, fruit clip and fruits of transgenic T3 homozygous plants, it was found that only 23 of the 200 Arabidopsis fruits were stained, and 177 Arabidopsis fruits showed no GUS signal, while GUS gene activity was detected in roots, stems, leaves and fruit clips (Fig. 2a–e). These results indicated that the specific promoter LFY in A. thaliana could not remove exogenous genes in roots, stems and leaves. Download Arabidopsis Motif Scanner latest version for Windows free. Arabidopsis Motif Scanner latest update: Ap Download Arabidopsis Motif Scanner latest version for Windows free. Arabidopsis Motif Scanner latest update: Ap

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The newly constructed expression vector with correct sequencing was named pCAMBIA-LFY-polseed-FLP. The E. coli plasmid was extracted according to the instruction manual of the plasmid extraction kit (Code No. 9760, TaKaRa, Dalian, China). The newly constructed pCAMBIA-LFY-polseed-FLP plant expression vector was transferred into A. tumefaciens strain EHA105 by heat shock (Huang et al. 2013), for subsequent genetic transformation of Arabidopsis and banana.Transformation and characterization of transgenic Arabidopsis plantsArabidopsis seeds were first sterilized with 70% ethanol for more than 10 s then sterilized with 10% sodium hypochlorite for 10 min, washed with sterile water 5 times and seeded on an MS solid medium (Clough and Bent 1998). The seeded medium was incubated at 4 °C and vernalized for 3 days and then shifted at 22 °C for 16 h light/8 h dark conditions. When A. thaliana had growth of 2 or 3 true leaves, they were transplanted to a seedling bowl with vermiculite as the substrate (vermiculite: vegetative soil 1:1) and continued to be cultured. An arched membrane was placed on the surface before culturing in an artificial climate incubator. During the vegetative growth phases, the daily light duration was 16 h (at 20001X) and the temperature was 20 °C. During the reproductive growth phase, the daily light duration was 16 h and the temperature was 20 °C. The light intensity was 20001X. The relative humidity of the incubator was between 70 and 90%. The seedlings were transplanted when they had growth of 4 to 6 true leaves, and were moisturized. Download Arabidopsis Motif Scanner latest version for Windows free. Arabidopsis Motif Scanner latest update: Ap