Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0...Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.展开更多
Objectives： Surgical repair of Achilles tendon （AT） rupture should immediately be followed by active tendon mobilization. The optimal time as to when the mobilization should begin is important yet controversial. Ea...Objectives： Surgical repair of Achilles tendon （AT） rupture should immediately be followed by active tendon mobilization. The optimal time as to when the mobilization should begin is important yet controversial. Early kinesitherapy leads to reduced rehabilitation period. However, an insight into the detailed mechanism of this process has not been gained. Proteomic technique can be used to separate and purify the proteins by differential expression profile which is related to the function of different proteins, but research in the area of proteomic analysis of AT 3 days after repair has not been studied so far. Methods： Forty-seven New Zealand white rabbits were randomized into 3 groups. Group A （immobilization group, n=l 6） received postoperative cast immobilization; Group B （early motion group, n= 16） received early active motion treatments immediately following the repair of AT rupture from tenotomy. Another 15 rabbits served as control group （Group C）. The AT samples were prepared 3 days following the microsurgery. The proteins were separated employing two- dimensional polyacrylamide gel electrophoresis （2D-PAGE）. PDQuest software version 8.0 was used to identify differentially expressed proteins, followed by peptide mass fingerprint （PMF） and tandem mass spectrum analysis, using the National Center for Biotechnology Information （NCBI） protein database retrieval and then for bioinformatics analysis. Results： Amean of 446.33,436.33 and 462.67 protein spots on Achilles tendon samples of 13 rabbits in Group A, 14 rabbits in Group B and 13 rabbits in Group C were suc- cessfully detected in the 2D-PAGE. There were 40, 36 and 79 unique proteins in Groups A, B and C respectively. Some differentially expressed proteins were enzyme with the gel, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry （MALDI-TOF MS）. We successfully identified 9 and 11 different proteins in Groups A and B, such as GAPDH, phosphoglycerate kinase 1, pro-alpha-1 type 1 collagen, peroxiredoxin 1, alpha-l-antiproteinase E a-1 and MAD2L1 binding protein, etc. And some with the molecular chaperone, oxidative stress, energy metabolism, signal transduetion, coupled with the tendon cell expression and protein synthesis, proliferate, differentiate and are closely related to the AT healing. The GAPDH protein was further validated through Western blotting. It was indicated that some differentially expressed proteins were involved in various metabolism pathways and may play an important role in initial healing of AT rupture. Conclusion： Differentially expressed proteins in rabbit healing AT model may contribute to 3 days healing of AT rupture through a new mechanobiological mechanism due to the application of postoperative early kinesitherapy.展开更多
文摘Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.
基金This study was financially supported by the National Natural Science Foundation of China （No. 30760256）.Acknowledgements We extend many thanks to Yang Fanyuan, Jin Hong, Yang Fenyin and Zhou Xinwen in Centre of Proteomics and Systems Biology, Institutes of Biomedical Sciences, Fudan University, Shanghai, China. We also thank Zhang Yuanting, Zhang Binyin and Yin Xiulian in Institutes of Biomedical Sciences, Qinghua University, Beijing, China for their help in Western blotting analysis.
文摘Objectives： Surgical repair of Achilles tendon （AT） rupture should immediately be followed by active tendon mobilization. The optimal time as to when the mobilization should begin is important yet controversial. Early kinesitherapy leads to reduced rehabilitation period. However, an insight into the detailed mechanism of this process has not been gained. Proteomic technique can be used to separate and purify the proteins by differential expression profile which is related to the function of different proteins, but research in the area of proteomic analysis of AT 3 days after repair has not been studied so far. Methods： Forty-seven New Zealand white rabbits were randomized into 3 groups. Group A （immobilization group, n=l 6） received postoperative cast immobilization; Group B （early motion group, n= 16） received early active motion treatments immediately following the repair of AT rupture from tenotomy. Another 15 rabbits served as control group （Group C）. The AT samples were prepared 3 days following the microsurgery. The proteins were separated employing two- dimensional polyacrylamide gel electrophoresis （2D-PAGE）. PDQuest software version 8.0 was used to identify differentially expressed proteins, followed by peptide mass fingerprint （PMF） and tandem mass spectrum analysis, using the National Center for Biotechnology Information （NCBI） protein database retrieval and then for bioinformatics analysis. Results： Amean of 446.33,436.33 and 462.67 protein spots on Achilles tendon samples of 13 rabbits in Group A, 14 rabbits in Group B and 13 rabbits in Group C were suc- cessfully detected in the 2D-PAGE. There were 40, 36 and 79 unique proteins in Groups A, B and C respectively. Some differentially expressed proteins were enzyme with the gel, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry （MALDI-TOF MS）. We successfully identified 9 and 11 different proteins in Groups A and B, such as GAPDH, phosphoglycerate kinase 1, pro-alpha-1 type 1 collagen, peroxiredoxin 1, alpha-l-antiproteinase E a-1 and MAD2L1 binding protein, etc. And some with the molecular chaperone, oxidative stress, energy metabolism, signal transduetion, coupled with the tendon cell expression and protein synthesis, proliferate, differentiate and are closely related to the AT healing. The GAPDH protein was further validated through Western blotting. It was indicated that some differentially expressed proteins were involved in various metabolism pathways and may play an important role in initial healing of AT rupture. Conclusion： Differentially expressed proteins in rabbit healing AT model may contribute to 3 days healing of AT rupture through a new mechanobiological mechanism due to the application of postoperative early kinesitherapy.
