El Hajj Mohammad, Hamdan Mohd Fahdli Bin, Avila Elena Martin, Day Anil
School of Biological Sciences, The University of Manchester, Manchester, UK.
Methods Mol Biol. 2018;1829:325-339. doi: 10.1007/978-1-4939-8654-5_22.
Plastid transformation is an attractive alternative to nuclear transformation enabling manipulation of native plastid genes and the insertion of foreign genes into plastids for applications in agriculture and industrial biotechnology. Transformation is achieved using dominant positive selection markers that confer resistance to antibiotics. The very high copy number of plastid DNA means that a prolonged selection step is required to obtain a uniform population of transgenic plastid genomes. Repair of mutant plastid genes with the corresponding functional allele allows selection based on restoration of the wild type phenotype. The use of deletion rather than point mutants avoids spontaneous reversion back to wild type. Combining antibiotic resistance markers with native plastid genes speeds up the attainment of homoplasmy and allows early transfer of transplastomic lines to soil where antibiotic selection is replaced by selection for photoautotrophic growth. Here we describe our method using the wild type rbcL gene as a plastid transformation marker to restore pigmentation and photosynthesis to a pale green heterotrophic rbcL mutant.
质体转化是核转化的一种有吸引力的替代方法，它能够操纵天然质体基因，并将外源基因插入质体，以应用于农业和工业生物技术领域。转化是通过使用赋予抗生素抗性的显性正向选择标记来实现的。质体DNA的极高拷贝数意味着需要一个延长的选择步骤来获得均匀的转基因质体基因组群体。用相应的功能等位基因修复突变质体质体基因允许基于野生型表型的恢复进行选择。使用缺失突变而非点突变可避免自发回复为野生型。将抗生素抗性标记与天然质体基因相结合可加速同质体的获得，并允许将转质体系早期转移到土壤中，在那里抗生素选择被光合自养生长选择所取代。在这里，我们描述了我们使用野生型rbcL基因作为质体转化标记的方法，以恢复浅绿色异养型rbcL突变体的色素沉着和光合作用。
