影响根癌农杆菌的电击转化条件

Studies on Electroporation Transformation of Agrobacterium tumefaciens

采用电击转化方法将双元载体质粒pCAM3300导入根癌农杆菌LBA4404,研究了不同实验条件对转化率的影响.结果表明:当电场强度为11 kV/cm时,转化率最高,每微克DNA得到9.8×103CFU;在细胞OD600为1.0时进行电转化,转化率最高,每微克DNA得到9.54×103CFU;在一定的细胞浓度范围内,电击转化率与细菌浓度密切相关,转化率随着细菌浓度的上升而上升;质粒大小与电击转化率之间没有明确的关系.将含pCAMBIA3300的根癌农杆菌与粗糙脉胞菌共培养转化,在含有膦化麦黄桐(PPT)(405 mg/L)的平板上筛选到转化子.PCR扩增转化子的染色体,初步证明Bar基因整合进粗糙脉胞菌的染色体中.

The binary vectors pCAM3300 was introduced into Agrobacterium tumefaciens by electroporation. Effects of different electroporation conditions on transformation frequency were studied. The results suggested transformation frequency reached to 9.8×10^3 CFU/μg DNA, the highest level, when the electrical field strength was 11kV/cm. Transformation frequency also reached a highest level of 9. 54×10^3 CFU/μg DNA when OD600 was at about 1.0. The transformation frequency was related to cell density, it increased with the increase of cell concentration. However, the size of plasmid had no specific relation with electroporation. The A. tumefaciens containing the binary vectors pCAM3300 co-cultured with Neurspora crassa and transformants were gained on plate containing 405mg/L of PPT. PCR results demonstrated that Bar gene was integrated into chromosome of Neurspora crassa.