农杆菌介导的水稻转Bt基因研究

Studies on Transformation of Bt Gene into Rice Mediated by Agrobacterium (Oryza sativa)

通过农杆菌介导法 ,将Bt毒蛋白基因的一种—cryIAb基因导入 2个云南水稻栽培品种(滇系 4号 ,合系 39号 )的愈伤组织中 ,经过潮霉素筛选后 ,获得了一批抗性苗 ,部分苗经PCR检测为阳性 ,GUS组织化学染色分析发现Bt基因已整合到水稻基因组 ,并由此建立了一套有效的水稻遗传转化体系。实验中发现培养器皿的透气性对水稻愈伤组织的形成、生长具有较大的影响 ,提高培养基的渗透势能够提高愈伤组织的分化率。选择合适的洗菌液渗透势能够大大降低洗菌对愈伤组织所造成的损伤 ,从而提高转化率。

CryIAb gene,one of Bt genes,was transferred into the calli of Yunnan rice-Dianxi No.4 and Hexi No.39 through agrobacterium associated method.Some hygromycin resistant (Hyg^+) transformants are regenerated after selection culture with hygromycin.some plantlets of Dianxi No.4 and Hexi No.39 were positive in PCR analysis,and some leaves of the plantlets show blue spots in GUS histochemical assay, which indicated that Bt gene was integrated into rice genome.An efficient transformation system was established by this study.In the experiment,we found that the ventilation of the container had great influence on the formation and growth of the calli.Osmotic potential of washing solution and media also affected transformation frequency,because they could reduce the death rate of the calli after washing treatmeat.