Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanke...Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.展开更多
Oilseed rape(Brassica napus L.)is one of the most important oil crops in the world.However,study on marker-free transgene of B.napus for bio-safety purpose is limited in this allotetraploid crop.In order to advance ma...Oilseed rape(Brassica napus L.)is one of the most important oil crops in the world.However,study on marker-free transgene of B.napus for bio-safety purpose is limited in this allotetraploid crop.In order to advance marker gene excision research,a newly designed Cre/lox system combining crossing and auto-excision strategy was introduced into B.napus.The system consists of 2 sets of independent vectors including pC35 Spro::T7 RP carrying T7 RNA polymerase and pCT7 pro::Cre carrying T7 promoter respectively.After hybridization of 2 according types of transgenic B.napus,marker gene would be removed as T7 RNA polymerase facilitate T7 promoter to promote Cre gene expression.Totally 52 and 46 positive To transgenic lines of these 2 vectors were obtained after identification by PCR and test trip.T1 plants from 3 T0 positive pC35 Spro::T7 RP lines and 2 T0 positive pC35 Spro::T7 RP lines were identified as single copy according to segregation ratio and were chosen for crossing.However,expression of CP4 EPSPS(glyphosate resistance gene)and OXY(bromoxynil resistance gene)were not found in F1 progeny,which proved that the excision was not complete.The possible reasons for our limited success were investigated and detailed analyses were performed.Although this system is not applicable for generating transgenic B.napus free from selectable marker gene,it provided valuable experience and clue for further improvement of this technique.Many other advantages and further improvement will be progressed in future work.展开更多
For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with ere gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-trans...For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with ere gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure. The transgenic plants were conformed by PCR analysis. The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome. Cloning and seguencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre//ox site-specif ic recombination .展开更多
基金the National Natural Science Foundation of China (30200185)the Science Foundation of Committee of Education of Chongqing Municipality,China (030208)
文摘Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.
基金supported by the National Program of Transgenic Variety Development of China(2018ZX08010-05B and 2019ZD080018)the Central Public-interest Scientific Institution Basal Research FundMajor Research Project of CAAS Science。
文摘Oilseed rape(Brassica napus L.)is one of the most important oil crops in the world.However,study on marker-free transgene of B.napus for bio-safety purpose is limited in this allotetraploid crop.In order to advance marker gene excision research,a newly designed Cre/lox system combining crossing and auto-excision strategy was introduced into B.napus.The system consists of 2 sets of independent vectors including pC35 Spro::T7 RP carrying T7 RNA polymerase and pCT7 pro::Cre carrying T7 promoter respectively.After hybridization of 2 according types of transgenic B.napus,marker gene would be removed as T7 RNA polymerase facilitate T7 promoter to promote Cre gene expression.Totally 52 and 46 positive To transgenic lines of these 2 vectors were obtained after identification by PCR and test trip.T1 plants from 3 T0 positive pC35 Spro::T7 RP lines and 2 T0 positive pC35 Spro::T7 RP lines were identified as single copy according to segregation ratio and were chosen for crossing.However,expression of CP4 EPSPS(glyphosate resistance gene)and OXY(bromoxynil resistance gene)were not found in F1 progeny,which proved that the excision was not complete.The possible reasons for our limited success were investigated and detailed analyses were performed.Although this system is not applicable for generating transgenic B.napus free from selectable marker gene,it provided valuable experience and clue for further improvement of this technique.Many other advantages and further improvement will be progressed in future work.
文摘For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with ere gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure. The transgenic plants were conformed by PCR analysis. The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome. Cloning and seguencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre//ox site-specif ic recombination .
