摘要
通过对EHA105菌株48 h不间断培养观察,绘制出其生长曲线图;同时用不同浓度的CaCl2溶液分别制备根癌农杆菌EHA105的感受态细胞,在转化过程中用不同的温度进行热激处理,统计分析阳性转化子个数,明确了采用冻融法将外源DNA导入农杆菌EHA105时的几个影响因素的参数。结果表明,当OD600值在1.0～1.5之间时,根癌农杆菌EHA105正处在对数生长期,活力最强;制备感受态细胞时,CaCl2溶液的浓度在6～10 mmol.L-1之间,无显著差异,但以10 mmol.L-1效果最佳,转化率达5.322×106.μg-1,热激处理温度在20～37℃之间均无显著差异,但37℃处理时转化率最高,达5.550×106.μg-1。
Growth of Agrobacterium tumefitciens EHA105, the quality of the competent cells and the heat-shock temperature during transformation are the three main factors affecting the transformation efficiency of A. tumefaciens EHA105. The growth of the strain EHA105 with 48 h continuous culture was observed based on which the growth curve was plotted. At the same time, different concentrations of CaCl2 and different temperatures in heatshocking during the transformation were tested in preparing the competent cells of EHA105. The number of positive transformants was determined and the factors, affecting introduction of exogenous DNA into the strain EHA105 by using the freeze-thaw method were identified. When the OD600 value ranged from 1. 0 to 1.5, the strain EHA105 was found at the logarithmic growth phase and had the highest vitality. No significant difference was observed in the transformation rates of competent cells prepared with the CaCl2 at the concentration of between 6 mmol ·L-1 and 10 mmol·L-1, but the best transformation rate was 5. 322 ×10^6·μg-1 at 10 mmol/L CaC12. No significant difference was found in transformation rates at the heat-shock temperatures from 20℃ to 37 ℃ during transformation, but the best transformation rate （5. 550 ×10^6·μg-1.） was achieved at 37 ℃. The results provide a reference for improving the tkahsformation efficiency of A. tumefaciens EHA105 competent cells.
出处
《热带生物学报》
2012年第1期22-27,31,共7页
Journal of Tropical Biology
基金
国家自然科学基金项目(30871581)
国家高技术研究发展"863"计划863项目(2007AA100701)
福建省科技厅国家科技项目(F2007AA100701)
现代农业产业技术体系建设专项资金项目(nycytx-024-01-06)
关键词
根癌农杆菌EHA105
生长曲线
冻融法
转化率
Agrobacterium tumefaciens EHA105
growth curve
freeze-thaw method
transformation rate
