优化白菜类蔬菜遗传转化体系的研究

Improvement of the transformation system in Chinese cabbage(Brassica campestris ssp. chinensis Makino)

采用农杆菌介导法将双元载体质粒pBI35S-AMF分别导入上海青"白菜和油青"菜心,对影响转化频率的抑菌剂和感染菌株等主要因素进行比较,优化了白菜类蔬菜的遗传转化体系.结果表明,在白菜类蔬菜的遗传转化体系中,选择氨苄青霉素作为转化农杆菌的抑菌剂为好,其抑菌浓度以300 mg/L为宜;预培养时间和共培养时间分别以3 d和2 d为适;LBA4404/pBI35S-AMF菌株的感染效率远高于EHA105/pBI35S-AMF.对KanR转化植株做进一步的分子检测结果显示,Southern印迹杂交阳性率为82.2%,转化频率达2.8%,且外源片段为多拷贝插入.多批次的转化结果证实,该体系的转化频率稳定.

The binary plasmid vector pBI35S-AMF was transferred into cv. Shanghaiqing of Chinese cabbage pak-choi （Brassica campestris ssp. Moteino chinensis var. communis Tsen et Lee） and cv. Youqing of flowering Chinese cabbage （B. campestris ssp. chinensis Makino var. parachinensis （Bailey） Tsen et Lee ） by Agrobacterium-mediated method. The pivotal factors, which influenced the transformation frequency, were compared, and the parameters of transformation system were optimized. It was showed that selective medium containing 300 mg/L ampicillin, which inhibited growth of Agrobacterium, led to production of more Kan^R shoots ; the time required for preculture and co-culture was three days and two days respectively; and the efficiency of inoculation with LBA4404/pBI35S-AMF was significantly higher than with EHA105/pBI35S-AMF. Molecular analysis to KanR seedlings showed that the frequency of Southern blotting was 82.2% and most of transformants were multi-copies integration. Moreover, the results indicated that the efficiency of transformation system was stable.