国槐遗传转化体系的优化

An effective Transformation System of Sophora japonica

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10min;侵染前的菌液和共培养基中添加200μmol·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)500mg·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 rain. Addition of 200μmol·L^-1 acetosyringone （AS） to the co-culture medium before infection significantly promoted S. japonica transformation. Antibiotic, 500 mgl· 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.