Staub J M, Maliga P
Waksman Institute, Rutgers, State University of New Jersey, Piscataway 08855-0759.
Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7468-72. doi: 10.1073/pnas.91.16.7468.
The plastid genome of higher plants is a circular double-stranded DNA molecule which is present in multiple identical copies. We report here an 868-bp plastid DNA minicircle, NICE1, that formed in tobacco (Nicotiana tabacum) plastids during transformation, as an unexpected product of homologous recombination. Such extrachromosomal elements are normally absent in plastids of higher plants. We have constructed shuttle plasmids containing NICE1 sequences which are maintained extrachromosomally when reintroduced into plastids by particle bombardment. Furthermore, recombination between homologous sequences in the shuttle plasmids and the main plastid genome occurs. Recombination products were characterized after recovery of the shuttle plasmids in Escherichia coli and of recombinant plastid genomes in the progeny of transformed plants. Our findings indicate that shuttle plasmids can be used to engineer plastid genes without concomitant integration of foreign DNA and to recover plastid mutations in E. coli.
高等植物的质体基因组是一个环状双链DNA分子，以多个相同拷贝的形式存在。我们在此报告一个868碱基对的质体DNA小环NICE1，它是在烟草（Nicotiana tabacum）质体转化过程中作为同源重组的意外产物形成的。这种染色体外元件在高等植物的质体中通常不存在。我们构建了含有NICE1序列的穿梭质粒，当通过粒子轰击将其重新导入质体时，这些质粒能以染色体外的形式维持。此外，穿梭质粒中的同源序列与主要质体基因组之间会发生重组。在大肠杆菌中回收穿梭质粒以及在转化植物后代中回收重组质体基因组后，对重组产物进行了表征。我们的研究结果表明，穿梭质粒可用于改造质体基因而无需伴随外源DNA的整合，并可在大肠杆菌中恢复质体突变。
