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利用双农杆菌/双质粒的共转化系统获得了无选择标记转pepc基因的水稻植株.采用两个农杆菌EHA105转化粳稻品种台粳9号幼胚诱导的胚性愈伤组织,其中一个农杆菌内的质粒表达载体的T-DNA区仅含有目的基因pepc,另一个的T-DNA区含有选择标记基因hpt和GUS报告基因.获得抗性愈伤的转化率为9.2%.85.3%的抗性愈伤分化成T0代株系,其中78.8%的T0代植株为GUS阳性转基因植株.PCR分析表明,在78个T0代GUS阳性植株(来源于23个抗性愈伤组织)中共有35个植株(来源于7个抗性愈伤组织)含有pepc基因,即在23个GUS阳性株系中发生共转化株系的频率为30.4%.7个共转化的株系中有5个株系自交产生后代植株中含有无标记转pepc基因植株,其比例为71.4%.无标记的转pepc基因水稻植株中PEPC活性平均比对照提高8.8倍;与非转基因对照植株相比,转pepc基因水稻植株表现出更强的光合能力.图5表3参26
Rice plants with pepc gene were selected by co-transformation system of Agrobacterium tumefaciens / double plasmid.The two Agrobacterium tumefaciens strains EHA105 and Agrobacterium tumefaciens were used to transform embryogenic callus induced by immature embryo of japonica rice cultivar, The T-DNA region of the plasmid expression vector contained only the target gene pepc and the other T-DNA region contained the selectable marker gene hpt and GUS reporter genes. The resistance to calli was 9.2% .85.3% resistance 78.0% of T0 plants were GUS-positive transgenic plants.PCR analysis showed that there were 35 plants in 78 T0 generation GUS-positive plants (derived from 23 resistant calli) (Derived from 7 resistant calluses) contained the pepc gene, ie 30.4% of the co-transformed lines occurred in 23 GUS-positive lines. Five of the seven co-transformed lines selfed The plants without progeny translocated with pepc gene accounted for 71.4% of the progeny plants.The average PEPC activity in unlabeled pepc transgenic rice plants was 8.8 times higher than that of the control plants. Compared with non-transgenic control plants, the pepc transgenic rice plants showed A stronger photosynthetic capacity