农杆菌介导的地黄遗传转化体系的优化

Optimization of Agrobacterium-mediated Transformation of Rehmannia glutinosa L.

旨在通过对农杆菌介导的地黄遗传转化体系影响因子的研究,探索最佳转化条件,为建立高效地黄遗传转化体系奠定基础。利用正交实验,通过检测地黄叶片中gus基因瞬时表达情况,对地黄遗传转化效率的决定因子进行了系统研究。正交试验结果表明,地黄遗传转化过程对转化效率影响程度从高到低的因素依次为:共培养时间、预培养时间、菌液浓度和侵染时间;以地黄叶片为外植体,材料不经过预培养,菌液OD600值为0.6,侵染时间5min,并在培养基中添加100μmol/L乙酰丁香酮,共培养3天条件下可达最高转化效率,gus基因瞬时表达率可达65.63%。通过对根癌农杆菌介导的地黄遗传转化体系的优化,建立了高效的瞬时表达转化体系。

This study aimed to improve the Agrobaeterium-mediated transformation system of Rehmannia glutinosa L. by optimizing the factors that influence the transformation efficiency. Through orthogonal experiment, conditions affecting Agrobacterium mediated transient transformation of leaf discs were evaluated, and the transient GUS assay was performed to observe the transformation efficiency. The results showed that, the co-cultivation time was the most significant factor influencing the transformation efficiency, followed by pre-cultivation time, bacteria concentration and infection incubation time. The optimized Agrobacterium- mediated transformation procedure for R. glutinosa was as follow： leaf discs without pre-cuhuring were infiltrated with A. tumefaciens for 5 rain at optical density （OD600） 0.6 in liquid MS medium, then the infected explants were co-cultivated with A. tumefaciens in shoot induction medium containing 100 μmol/L acetosyringone in dark for 3 days. Under optimal conditions, the highest transient expression efficiency of GUS was 65.63%. An effective Agrobaeterium-mediated transformation system was established for R. glutinosa by optimizing the influencing factors.