水稻转化体系的改进及转os-miR398基因植株的获得

针对根癌农杆菌介导的愈伤转化技术在实际应用中转化效率低及农杆菌污染等问题,对该方法在水稻转化过程进行改良:(1)在转化前将空白愈伤从培养基取出,于室温放置在空白皿中约24 h,使之处于饥饿状态,以利于T-DNA转化并提高转化率;(2)在愈伤与农杆菌共培养并经无菌洗脱后,在转移到相应培养基之前,将其于室温下继续放置在含有滤纸的培养皿里约24 h,从而有效地抑制农杆菌生长.采用本改良措施,成功将所克隆构建的os-miR398(水稻microRNA398)前体基因表达载体转化入水稻,与对照相比,改良后水稻转化效率可提高10%.

Optimization in Rice Transformation System and Regeneration of Transgenic Plants with os-miR398 Gene

The system of rice callus transformation by Agrobacterium tumefaciens is a prevalent and mature approach of transgenic rice.However,there are some shortage such as the low frequency of transformation and severe degree of pollution by A.tumefaciens in reality.To resolve those problems we optimize the protocol of rice transformation by two pathways.Firstly,to increase the frequency of transformation,we placed the rice calli from culture medium on blank utensil about 24 hours before transformation so that the calli were on a hungered state and was easy to absorb T-DNA.Secondly,to inhibit the pollution by A.tumefaciens,we keep on placed the calli on blank utensil about 24 hours after co-incubating calli with A.tumefaciens and washing A.tumefaciens by sterile water subsequently so that suppress the growth of A.tumefaciens.Based on this way,we successfully transformed the expressional plasmid which was been constructed with the precursors DNA of os-miR398 gene into rice.The result showed that optimized approach could increase frequency of transformation 10% and suppress the growth of A.tumefaciens one decuple compare to control.