Kharazmi Mitra, Hammes Walter P, Hertel Christian
Institute of Food Technology, University of Hohenheim, Stuttgart, Germany.
Syst Appl Microbiol. 2002 Dec;25(4):471-7. doi: 10.1078/07232020260517580.
A marker rescue system based on the repair of the kanamycin resistance gene nptII was constructed for use in Gram-positive bacteria and established in Bacillus subtilis 168. Marker rescue was detected in vitro using different types of donor DNA containing intact nptII. The efficiency of marker rescue using chromosomal DNA of E. coli Sure as well as plasmids pMR2 or pSR8-30 ranged from 3.8 x 10(-8) to 1.5 x 10(-9) transformants per nptII gene. Low efficiencies of ca. 10(-12) were obtained with PCR fragments of 792 bp obtained from chromosomal DNA of E. coli Sure or DNA from a transgenic potato. B. subtilis developed competence during growth in milk and chocolate milk, and marker rescue transformation was detected with frequencies of ca. 10(-6) and 10(-8), respectively, using chromosomal DNA of E. coli Sure as donor DNA. Although the copy number of nptII genes of the plant DNA exceeded that of chromosomal E. coli DNA in the marker rescue experiments, a transfer of DNA from the transgenic plant to B. subtilis was detectable neither in vitro nor in situ.
构建了一种基于卡那霉素抗性基因nptII修复的标记拯救系统,用于革兰氏阳性菌,并在枯草芽孢杆菌168中得以确立。使用含有完整nptII的不同类型供体DNA在体外检测标记拯救。使用大肠杆菌Sure的染色体DNA以及质粒pMR2或pSR8 - 30进行标记拯救的效率为每nptII基因3.8×10(-8)至1.5×10(-9)个转化体。从大肠杆菌Sure染色体DNA或转基因马铃薯DNA获得的792 bp PCR片段的效率约为10(-12)。枯草芽孢杆菌在牛奶和巧克力牛奶中生长时会形成感受态,分别使用大肠杆菌Sure的染色体DNA作为供体DNA,以约10(-6)和10(-8)的频率检测到标记拯救转化。尽管在标记拯救实验中植物DNA的nptII基因拷贝数超过了大肠杆菌染色体DNA的拷贝数,但在体外和原位均未检测到DNA从转基因植物转移到枯草芽孢杆菌。