Efficient targeting of foreign genes into the tobacco plastid genome.
Efficient targeting of foreign genes into the tobacco plastid genome.
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DOI：
10.1093/nar/22.19.3819
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发表时间：
1994-09
影响因子：
14.9
通讯作者：
O. Zoubenko;Lori A. Allison;Z. Sváb;P. Maliga
中科院分区：
文献类型：
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作者：
O. Zoubenko;Lori A. Allison;Z. Sváb;P. Maliga
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。该载体基于在E.而质体中没有。壮观霉素抗性（aadA）基因和多克隆位点（MCS）的侧翼是1.8-kb和1.2-kb ptDNA序列。载体DNA的生物射弹递送，接着大观霉素选择，以可再现的频率产生质体转化体，每个轰击样品约1个转质体系。选择的aadA基因和克隆到MCS中的连接的非选择性基因通过两个同源重组事件经由侧翼ptDNA序列并入ptDNA中。由此产生的转质体是稳定的，并通过母体传递给种子后代。pPRV载体系列靶向在趋异转录的trnV基因和rps 12/7操纵子之间的插入。适当定向的转基因的通读转录的缺乏使得载体成为研究转基因启动子活性的理想选择。
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.