农杆菌介导粳稻高效转化体系的研究

Study on Efficient Agrobacterium-mediated Transformation System on Japonica Rice

以5个粳稻品种为材料,研究农杆菌介导转化中影响粳稻转化的几个重要因素。通过研究发现,根癌农杆菌菌株EHA105的转化率明显高于LBA4404,根癌农杆菌菌株EHA105介导的水稻转化率为25.3%,而LBA4404为7.6%;不同外植体的诱导率、转化率和分化率存在明显差异,以幼胚为外植体材料,它的出愈率和转化率明显比成熟胚高,而分化率则明显低于成熟胚;不同水稻品种在转化率与分化率上差异也很明显,反映了不同基因型水稻品种的差异,同时研究发现同一品种不同批次在转化率和再生频率上差异比较显著,反映了在水稻转化过程中还存在一些未知条件对于转化的影响;通过对抗性愈伤和转化植株的叶片、根的GUS染色,证明GUS基因在不同组织中表达;并且通过PCR和Southern杂交分子分析,发现所检测的植株均为转基因植株,足以证明这个高效转化体系的可靠性。

A few of important factors have been investigated on influencing the frequency of rice Agrobacterium-mediated transformation by using 5 Japonica cultivars. The research showed that Agrobacterium tumefaciens strain EHA105 was more efficient than that of LBA 4404 for rice transformation, and it's transformation frequency was 25.3 percent while LBA 4404's transformation frequency was 7.6 percent. Main difference was found in frequency of inducement, transformation, differentiation of different explants and cultivars. Frequency of inducement and transformation from young embryos was obviously higher than that of mature embryos, but it′s differentiation frequency was lower than that of mature embryos. Meanwhile, frequency of transformation, differentiation of different cultivars was different, which indicated rice genotype difference. And frequency of transformation, differentiation of different batches from the same cultivar was different, which showed that a few of factors influencing transformation were not yet found in rice transformation. It was proved by GUS gene expression through GUS stain of resistant callus, leaves and roots of transformation plants. Transgenic rice was testified by analysis of PCR and Southern-blotting. It showed that transformation system was reliable for Japonica rice.