超声波辅助农杆菌介导八棱海棠转rolC基因

Transformation of rolC gene to Maius robusta by SAAT

应用超声波辅助农杆菌介导法,对八棱海棠进行rolC基因转化,以期提高转化效率并获得转基因植株。利用gus基因瞬间表达的方法研究了超声波处理时间、处理时期和农杆菌悬浮液中乙酰丁香酮(As)浓度对rolC基因转化率的影响。结果表明,叶盘在D600nm为0．6且含有75 mg／L As的农杆菌悬浮液中侵染2 min后,用功率为100 W的超声波处理30 s,再浸泡2．5 min,然后放到再生培养基上共培养3 d,能获得最佳的gus基因瞬间表达率。最佳处理条件下转化683枚八棱海棠叶片,共得到138个抗性愈伤组织和15株抗性苗,转化率为2．2％。GUS染色、PCR及Southern blotting检测结果显示,有12个八棱海棠株系的基因组中整合了完整的外源rolC基因。

Malus robusta Rehd. was transformed with rolC gene by SAAT （Sonication assisted Agrobacterium-mediated transformation） in order to improve transformation efficiency and get transgenie shoots. The factors, which influenced the transformation efficiency, such as sonieation treatment time, stage and the concentration of Agrobacterium suspension, were all investigated via gus staining instant expression. The results showed that when the leaves were treated for 30 seconds with 100 W power sonieation after immersed in Agrobacterium suspension （D600nm = 0.6 ）containing 75 mg/L Aeetosyringone （As） for 2 minutes , then immersed for 2.5 minutes sequentially,at last placed in regeneration medium for 3-day co-culture could get the highest 81.5% gus transient expression. Total 683 leaves were transferred under the optimum treatment condition, then 138 resistant callus and 15 resistant shoots were obtained （transformation rate 2.2%）. After verified by GUS staining, PCR amplification and Southern blotting, it was proved that the rolC gene had been integrated into genome of 12 resistant shoots.