采用根癌农杆菌介导的国槐叶盘转化法,在建立修饰的豇豆胰蛋白酶抑制剂基因(sck)转化体系过程中,对影响农杆菌转化频率的各种因素进行研究。结果表明: 国槐叶片需在黑暗条件下在MS培养基上预培养5天,农杆菌菌株选用GV3101,农杆菌共培养3天较合适; 农杆菌菌液浓度OD600约为0.7,感染时间10 min; 侵染前的菌液和共培养基中添加200 μmol·L-1乙酰丁香酮(AS)对国槐遗传转化有明显的促进作用; 抑制农杆菌生长的抗生素浓度以头孢霉素(Cef)500?mg·L-1最好。PCR和Southern blotting检测证实 sck基因已整合到国槐基因组中。
This paper investigated several factors affecting transformation frequency of Sophora japonica in establishing the modified sck gene transfer system process with Agrobacterium tumefaciens-mediated leaf disc transformation method. The results showed that S. japonica leaves were required to pre-culture on MS medium for 5 d in the dark. Agrobacterium strains, GV3101, were selected, and the Agrobacterium was needed to co-cultivation for 3 d. Agrobacterium bacterium concentration was about 0.7 in OD600, and the infection time was 10 min. Addition of 200μmol·L-1 acetosyringone (AS) to the co-culture medium before infection significantly promoted S. japonica transformation. Antibiotic, 500 mg·L-1 Cef, was best for inhibiting Agrobacterium growth. PCR and Southern blotting confirmed that sck gene had been integrated into the S. japonica genome.
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