摘要
目的探索根癌农杆菌的高效转化方法。方法考察质粒经电击转化法转化根癌农杆菌 (A grobacteriumtumefaciensLBA440 4)的影响因素 ,并优化其转化条件 ;研究采用三亲杂交方法 ,将带有vgb基因簇的重组质粒 pYG3 0 8导入A .tumefaciensLBA440 4;经PCR扩增反应进行验证。 结果外加电场强度和脉冲时间是决定电转化效率的关键因素 ,两者适当组合才可获得高转化效率 ;采用对数期中期的细菌和提高细菌浓度均可提高转化效率。用三亲杂交方法转化A .tumefaciensLBA440 4,可得到较高的转化效率。所获抗性转化子的质粒DNA酶切图谱正确。结论带有双元载体 (含vgb基因 )
Objective To explore effective transformation methods for Agrobacterium tumefaciens LBA4404.Methods Electroporation was performed to transfer plasmid DNA of pYG308 into A.tumefaciens LBA4404.A number of factors that affected transformation efficiency were investigated.The plasmid pYG308 harboring the functional vgb gene cluster was transformed by tri-parental mating into A.tumefaciens LBA4404.The PCR amplification of vgb was conducted.Results Optimized electroporation protocol that yielded highly efficient gene transfer in A.tumefaciens LBA4404.High transformation efficiency was obtained with an appropriate combination of field strength and pulse time.Transformation efficiency could also be elevated by using the host cell harvested in the middle exponential growth phase and increasing the density of the host cells.Two plasmids were isolated from the transformants growing on the LB plate (containing Rif100,Str300,Km50),and shown to have the right physical map.Conclusions These evidence and PCR amplification of vgb gene demonstrate the successful construction of a bi-plasmid recombinant strain.
出处
《沈阳药科大学学报》
CAS
CSCD
2003年第6期451-454,共4页
Journal of Shenyang Pharmaceutical University
基金
国家高技术研究发展计划资助项目 (863计划 2 0 0 1AA2 1 42 0 1 )
