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采用GUS基因瞬时表达检测方法,通过正交试验以AS浓度、侵染菌液OD值、侵染时间、共培养时间和恢复培养时间5个因素在4个水平上进行分析,优化了农杆菌介导的大豆胚尖遗传转化体系,并在此基础上进行了抗逆基因GmPK的遗传转化。结果表明,采用共培养培养基中添加100μmol/L AS、侵染菌液OD600值0.9、侵染15h、共培养5d和恢复培养3d的转化条件最佳,GUS阳性率达74.59%,经PCR及RT-PCR进一步验证获得了转基因阳性植株。利用优化的最佳条件进行抗逆基因GmPK的转化,炼苗移栽成活的再生植株经PCR及PCR-Southern blotting验证,初步证明外源基因已经整合至大豆基因组,转化率为0.6%。
Using GUS gene transient expression detection method, four factors including AS concentration, OD value of infecting bacteria, infection time, co-culture time and recovery culture time were analyzed by orthogonal test, and Agrobacterium mediated Guided soybean embryo genetic transformation system, and on the basis of the genetic transformation of the resistance gene GmPK. The results showed that the optimum conditions for the transformation were as follows: adding 100μmol / L AS into the culture medium, OD600 value 0.9, infecting 15h, co-culture for 5 days and recovery culture for 3d, the positive rate of GUS was 74.59% RT-PCR further verified transgenic positive plants. The optimal conditions were optimized for the transformation of GmPK, and the regenerated plantlets were harvested and transformed into soybean genome by PCR and PCR-Southern blotting. The transformation rate was 0.6%.