[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF wa...[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.展开更多
We have developed a 'double T-DNA' binary vector system for generating selectable marker-free transgenic plants by Agrobacterium-mediated transformation. The 'double T-DNA' binary vector pDLBRBbarm whi...We have developed a 'double T-DNA' binary vector system for generating selectable marker-free transgenic plants by Agrobacterium-mediated transformation. The 'double T-DNA' binary vector pDLBRBbarm which carried two independent T-DNAs, one containing a selectable marker neomycin phosphotransferase (nptII) gene and the other a bargene, was constructed. Transgenic tobacco (Nicotiana tabacum L.) plants were then produced by Agrobacterium-mediated transformation with this vector. Frequency of the primary transformants co-integrated with npt II gene and bar gene was 59.2%. Segregation of two T-DNA regions was found in 3 out of 4 T-1 lines from co-transformed T-0 plants with nptII and bar PPT-resistant and kanamycin-sensitive plants were in approximate 19.5% of the T-1 plants. The result indicated that this 'double T-DNA' vector system could be a workable approach to generate transgenic plants free from selectable marker genes. Co-transformation of nptII gene and bar gene to plants with mixtures of Agrobacterium tumefaciens strains containing single T-DNA vectors was also tested. Frequency of co-transformed plants was 20.0%-47.7% and relatively low as compared with that of 'double T-DNA' vector system.展开更多
Exoplants of tomato(Lycopersicon esculentum) leaf were transformed with Ti plasmids using binary vector system.After screening.with selection culture, kanamycin-resistant seedling were obtained from callus. Molecular ...Exoplants of tomato(Lycopersicon esculentum) leaf were transformed with Ti plasmids using binary vector system.After screening.with selection culture, kanamycin-resistant seedling were obtained from callus. Molecular hybridization proved the integration of Km gene into plant cell genome via A.tumefaciens. Higher activity of Nos-NPTase was demonstrated in the transformed plant,thus confirming the successful expression of the resistance gene in recipient cells.展开更多
基金Supported by Bioreactor Important Special Item of 863-Program inthe "Eleventh Five-Year" Plan (No. 2007AA100503)Science and Technology Development Key Plan of Jilin Province( No.20070922)+1 种基金Cultivation Fund of Scientific and Technical Innovation Project Major Program of Higher Education Institutions ( No.70S018)Science and Technology Plan of Changchun City (No.06GG150)~~
文摘[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.
文摘We have developed a 'double T-DNA' binary vector system for generating selectable marker-free transgenic plants by Agrobacterium-mediated transformation. The 'double T-DNA' binary vector pDLBRBbarm which carried two independent T-DNAs, one containing a selectable marker neomycin phosphotransferase (nptII) gene and the other a bargene, was constructed. Transgenic tobacco (Nicotiana tabacum L.) plants were then produced by Agrobacterium-mediated transformation with this vector. Frequency of the primary transformants co-integrated with npt II gene and bar gene was 59.2%. Segregation of two T-DNA regions was found in 3 out of 4 T-1 lines from co-transformed T-0 plants with nptII and bar PPT-resistant and kanamycin-sensitive plants were in approximate 19.5% of the T-1 plants. The result indicated that this 'double T-DNA' vector system could be a workable approach to generate transgenic plants free from selectable marker genes. Co-transformation of nptII gene and bar gene to plants with mixtures of Agrobacterium tumefaciens strains containing single T-DNA vectors was also tested. Frequency of co-transformed plants was 20.0%-47.7% and relatively low as compared with that of 'double T-DNA' vector system.
文摘Exoplants of tomato(Lycopersicon esculentum) leaf were transformed with Ti plasmids using binary vector system.After screening.with selection culture, kanamycin-resistant seedling were obtained from callus. Molecular hybridization proved the integration of Km gene into plant cell genome via A.tumefaciens. Higher activity of Nos-NPTase was demonstrated in the transformed plant,thus confirming the successful expression of the resistance gene in recipient cells.
