根癌农杆菌转化条件优化的研究

Study on the Optimization of Transformation Conditions for Agrobacterium tumefaciens

目的:确立一个快速高效转化根癌农杆菌的实验方案。方法:以pCAMBIA1305.1质粒作为外源DNA转化农杆菌GV3101。通过对GV3101生长状态的测定,并对重悬液的种类和浓度、速冻时间、热处理温度等条件逐一进行筛选,以确定最适合GV3101转化的实验条件。结果:以TSS缓冲液重悬细胞,液氮速冻3min后于28℃热处理5min,农杆菌转化效率可达到105。结论:改进的转化方法转化率高,重复性好,简单易行,为用农杆菌介导法进行植物遗传转化的第一个限速步骤提供了很好的解决方案。

Objective：To establish a high efficient protocol for transformation of Agrobacerium tumefaciens. Methods： Agrobacerium tumefaciens GV3101 was transformed by the plasmid pCAMBIA1305.1. To define the best experimental conditions for transformation of A. tumefaciens, the growth rate of the A. tumefaciens GV3101 was measured, and the sort and concentration of resuspension solution, freezing time and the thawing temperature were tested. Results： By resuspended inTSS buffer, frozen in liquid nitrogen for3 rain and thawed in 28℃ for5 min, the efficiency of transformation could be improved to 10℃ . Conclusion： The modified protocol of transformating A. tumefaciens is efficient, simple and easily repeated. It proposed a good resolution for the first key step of transformation of plant via A. tumefaciens.