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本研究以马铃薯(Solanum tuberosum L.)试管薯作为转化受体,利用农杆菌介导法将从梭梭中获得的甜菜碱醛脱氢酶基因(HaBADH)转化马铃薯,采用正交试验设计优化了影响马铃薯转化的4个主要因子,建立了农杆菌介导的马铃薯高效转化体系,获得了转基因植株。研究结果表明,转化过程中,浸染时间15 min,菌液浓度OD600为1.0,筛选培养时卡那霉素浓度30 mg/L、品种为大西洋为最佳转化方案,抗性芽分化率最高达到了35.9%;4个因子中基因型对转化效率影响最大,菌液浓度的影响次之,卡那霉素浓度和浸染时间的影响最小。经PCR、RT-PCR及Southern blot检测,证明了HaBADH基因整合到马铃薯中,阳性转化率为45.3%。
In this study, potato (Solanum tuberosum L.) tube tubers as a transformation receptor, the use of Agrobacterium-mediated transformation obtained from the Haloxylon amylindoline aldehyde dehydrogenase gene (HaBADH) transformed potato, using orthogonal design to optimize Four main factors affecting potato transformation were established. Agrobacterium-mediated efficient potato transformation system was established and transgenic plants were obtained. The results showed that in the transformation process, the dipping time was 15 min, the OD600 of the bacterial liquid was 1.0, the concentration of kanamycin was 30 mg / L when the culture was selected, and the best transformant was the Atlantic, the highest rate of resistant bud differentiation reached 35.9%. The genotypes of four factors had the greatest effect on the transformation efficiency, followed by the concentration of the bacterial liquid, and the least influence of the kanamycin concentration and the immersion time. The results of PCR, RT-PCR and Southern blot showed that the HaBADH gene was integrated into potato and the positive conversion rate was 45.3%.