叶盘共培养法介导的基因转移与两种转基因植物的鉴定

Leaf—disc Coculture Mediated Gene Transfer and the Expression and Identification of Foreign Genes in Ttansgenic Plants

本文以共整合型Ti质粒载体pGV3850:1103neo中pNOS-NPTⅡ(Km^R)嵌合基因为遗传标志,应用改良的叶盘共培养法对烟草和番茄进行了遗传转化。通过施加选择压力,筛选出具有Km^R表型的愈伤组织,并由此获得转基因烟草和番茄植株。通过胭脂碱的高压电泳检测和生物素探针的尼龙膜印迹杂交,分别从侧面(间接)和正面(直接)证实了Km^R基因在上述两种转化细胞中的整合和表达。还探讨了转化、培养和筛选过程中的一些细节,并对改进技术的原理及其效果进行了讨论。

Transgenic tobacco(Nicotiana tabacum)and tomato(Lycopersicon esculentum) plants, which express chimeric pNOS—NPT Ⅱ genes derived from Ti plasmid and TN5, respectively, were obtained in our experiments. All of the transgenic plants regenerated from transformed cells by using the improved cocultivation methods of leaf disks with Agrobacterium tumefaciens C58C1Rif^R(pGV3850::1103neo), subsequently the transformed cells were cultured in vitro and went through the whole processes of somatic embryogenesis. The integration, expression and transmission of foreign genes of these transgenic plants were confirmed by growing on the selecting medium supplemented with kanamycin(100μg/ml), induction of the division and the morphogenesis of the resistant cells, detection of nopaline synthesized by the nopaline synthase of the product of an cointegrated marker gene nos with Km^R gene, and by dot-blot hybridization of biotin-labeled probe with target DNAs from transgenic plants. Some details of the techniques for transformation, selection and subculture were discussed.