农杆菌介导的大豆成熟胚芽尖遗传转化体系的优化

OPTIMIZATION OF AGROBACTERIUM-MEDIATED TRANSFORMATION SYSTEMS OF SOYBEAN MATURE EMBRYONIC TIP

以大豆品种‘浙春3号’的胚芽尖为受体材料,利用农杆菌介导法转化抗逆相关大豆转录因子GmGT-2A,为探讨过量表达该基因以提高大豆对非生物胁迫的抗逆性准备材料,通过研究预培养时间、共培养培养基的6-BA浓度、侵染时间等因子对转化效率的影响,系统优化了大豆的成熟胚芽尖转化体系。单因素优化实验结果表明:预培养时间、6-BA浓度和侵染时间均对大豆遗传转化有显著的影响,三者各自最优条件分别为胚芽尖预培养时间1.5d;共培养基6-BA浓度8mg/L和浸染时间10h。根据以上结果设计三因素三水平正交试验对转化体系进行系统优化,结果表明:以上因素对转化效率的影响程度从大到小依次为:侵染时间、预培养时间和6-BA浓度;最佳转化体系中各因素水平与单因素优化结果一致。本研究获得的最高平均转化效率为5.67%。实验还初步探讨了IBA浓度对再生苗生根的影响,结果表明在生根培养基中不加入IBA比加入IBA时生根时间短,且生根数量多,移栽易成活。利用上述方法得到的抗性苗,经目的基因、标记基因和构建特异性的PCR检测,表明目的基因已成功导入到大豆基因组中。

Transcription factor gene GmGT-2A was introduced into soybean variety Zhechun 3 via Agrobacterium-mediated soybean mature embryonic tip transformation systems.The effects of three main factors on the transformation efficiency,including pre-culture time of embryonic tips,6-BA concentration in the co-culture medium and the infection time were studied.Single factor optimizing test showed that transformation efficiency was affected by all the three factors.The optimal levels for each factor was 1.5d for pre-culture time,10h for infection time and 8mg/L for 6-BA concentration in the co-culture medium.Results of further orthogonal test showed that these three factors could be ranked as infection time,pre-culture time,and 6-BA concentration according to their effectiveness on transformation efficiency.The optimal transformation system at the same levels for each factor as mentioned above resulted in a highest transformation efficiency of 5.67%.A preliminary test also showed that rooting medium without IBA addition was better than that with 0.2mg/L IBA in terms of root emerging time and roots qualities.The transformation events were confirmed using target gene-specific,selective marker gene-specific and construction-specific PCR analysis.