Maliga P, Carrer H, Kanevski I, Staub J, Svab Z
Waksman Institute, Rutgers, State University of New Jersey, Piscataway 08855-0759.
Philos Trans R Soc Lond B Biol Sci. 1993 Nov 29;342(1301):203-8. doi: 10.1098/rstb.1993.0148.
We have developed efficient transformation protocols to modify each of the 500-10,000 plastid genome copies in a tobacco cell. The transforming DNA is introduced on the surface of microscopic tungsten particles by the biolistic process. Selection for transplastomes is by spectinomycin resistance based on expression of aminoglycoside-3"-adenyltransferase from a chimeric aadA gene in the transforming DNA. Manipulations that are now feasible include replacement of endogenous plastid genes with DNA sequences modified in vitro, targeted gene disruption, and insertion of reporter genes into the plastid genome. Alternative methods for plastid genome manipulations may be developed utilizing an extrachromosomal element which was identified during the transformation studies. Introduction of foreign genes under control of plastid gene expression elements results in duplication of endogenous regulatory sequences. A sensitive somatic assay to detect deletions via such direct repeats confirmed that these sequence duplications do not result in significant genome instability. The ability to transform plastids will facilitate the study of plastid gene regulation, and the application of genetic engineering to crop improvement.
我们已经开发出高效的转化方案，用于修饰烟草细胞中500至10000个质体基因组拷贝中的每一个。通过生物弹道过程将转化DNA引入微观钨颗粒表面。基于转化DNA中嵌合aadA基因的氨基糖苷-3''-腺苷转移酶的表达，通过壮观霉素抗性选择转质体基因组。现在可行的操作包括用体外修饰的DNA序列替换内源质体基因、靶向基因破坏以及将报告基因插入质体基因组。利用在转化研究中鉴定出的一种染色体外元件，可能会开发出质体基因组操作的替代方法。在质体基因表达元件控制下引入外源基因会导致内源调控序列的重复。一种通过这种直接重复检测缺失的灵敏体细胞检测方法证实，这些序列重复不会导致显著的基因组不稳定。质体转化能力将有助于质体基因调控的研究，以及基因工程在作物改良中的应用。
