花生子叶节外植体遗传转化体系的建立

摘要

为了建立花生的高效遗传转化体系,利用花生子叶节高效再生体系,以农杆菌介导法,用含有表达质粒pBOG3VP7的根癌农杆菌进行外源基因转化,优化花生的遗传转化体系.通过对影响农杆菌介导的花生遗传转化的诸多因素进行的优化,试验表明:侵染菌液添加100 μmol/L AS,结合负压处理,农杆菌侵染10 min,共培养3d,延后选择2w.对抗性植株进行PCR和PCR-Southern检测,11株中有6株PCR反应呈阳性,其中有3株Southern杂交有特异性目标带出现.结果表明,外源基因已经整合到了花生基因组上.%In order to establish efficient genetic transformation system of peanuts, applying to regeneration system of peanut cotyledon node explants, the colony of LBA4404 containing pBOG3VP7 was used for thransformation.Genetic transformation system of peanut cotyledon nodes via Agrobacterium tumefaciens-mediated was developed, in which the effects of several factors influencing the tranformation were investigated and optimized.Peanut cotyledon nodes were infected under the treatment of negative pressure for 10 min, co-cultured for 3 days, and regenerative shoots were screened on selective medium after sub-culture of 2 weeks.Concentration of AS with 100 μmol/L was helpful for successful infection.6 plants among 11 plants which can grow up through selection on medium containing 125 mg· L-1 Kanamycin were assessed by PCR amplification.Then the genomie DNA of PCR positive plants were further analyzed with PCR-Southern blot hybridizations.3 plants had all DNA blotting band.The results show that the foreign gene has been integrated into genome of transformated peanuts.