Zoubenko O V, Allison L A, Svab Z, Maliga P
Waksman Institute, Rutgers, State University of New Jersey, Piscataway 08855-0759.
Nucleic Acids Res. 1994 Sep 25;22(19):3819-24. doi: 10.1093/nar/22.19.3819.
The pPRV plasmids are vectors for targeted insertion of foreign genes into the tobacco plastid genome (ptDNA). The vectors are based on the pUC119 plasmid which replicates in E. coli but not in plastids. The spectinomycin resistance (aadA) gene and a multiple cloning site (MCS) are flanked by 1.8-kb and 1.2-kb ptDNA sequences. Biolistic delivery of vector DNA, followed by spectinomycin selection, yields plastid transformants at a reproducible frequency, approximately 1 transplastomic line per bombarded sample. The selected aadA gene and linked non-selectable genes cloned into the MCS are incorporated into the ptDNA by two homologous recombination events via the flanking ptDNA sequences. The transplastomes thus generated are stable, and are maternally transmitted to the seed progeny. The pPRV vector series targets insertions between the divergently transcribed trnV gene and the rps12/7 operon. The lack of readthrough transcription of appropriately oriented transgenes makes the vectors an ideal choice for the study of transgene promoter activity.
pPRV质粒是用于将外源基因靶向插入烟草质体基因组（ptDNA）的载体。这些载体基于在大肠杆菌中复制但不在质体中复制的pUC119质粒。壮观霉素抗性（aadA）基因和多克隆位点（MCS）两侧分别是1.8 kb和1.2 kb的ptDNA序列。通过基因枪介导载体DNA传递，随后进行壮观霉素筛选，可产生频率可重复的质体转化体，每个轰击样品约有1个转质体系。通过侧翼ptDNA序列的两次同源重组事件，将克隆到MCS中的所选aadA基因和相关的非选择基因整合到ptDNA中。由此产生的转质体基因组是稳定的，并通过母系遗传给种子后代。pPRV载体系列靶向插入反向转录的trnV基因和rps12/7操纵子之间。适当定向的转基因缺乏通读转录，使得这些载体成为研究转基因启动子活性的理想选择。
