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
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.
