High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene.

We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops. IMAGES:

本研究报道，相较于采用突变16S核糖体RNA（16S rRNA）基因作为筛选标记的策略，通过编码氨基糖苷类3"-腺苷酰转移酶（aminoglycoside 3"-adenylyltransferase）的嵌合aadA基因进行筛选，烟草的质体转化（plastid transformation）频率提升了100倍。aadA基因的表达可赋予宿主对壮观霉素（spectinomycin）与链霉素（streptomycin）的抗性。在以质粒pZS197开展转化实验时，嵌合aadA基因被克隆至rbcL质体基因序列与开放阅读框ORF512质体基因序列之间。将pZS197 DNA通过基因枪法（biolistic delivery）导入烟草叶肉细胞后，以壮观霉素抗性作为筛选条件。DNA凝胶印迹（DNA gel-blot）分析证实，嵌合aadA基因通过两侧质体基因序列介导的两次同源重组事件，成功整合至质体基因组中。该嵌合基因在受体细胞中形成同质体（homoplasmic）状态，并可稳定传递至母系种子子代。实现陆生植物质体的常规转化的技术能力，为操控光合作用过程以及向作物质体基因组中引入外源基因开辟了全新路径。图像资料：