College of Life Science, Hunan Normal University, State Key Laboratory Breeding Base of Microbial Molecular Biology, Changsha, 410081, People's Republic of China,
Biotechnol Lett. 2013 Dec;35(12):2177-83. doi: 10.1007/s10529-013-1310-7. Epub 2013 Aug 24.
Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T1 progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops.
利用组织特异性调控 Cre/loxP 系统创建了无标记、基因修饰的水稻，该系统中 Cre 重组酶基因和潮霉素磷酸转移酶基因（hpt）被两个直接定向的 loxP 位点包围。Cre 表达受花特异性启动子 OsMADS45 或种子特异性 napin 激活，导致重组酶和标记基因的同时切除。通过 T1 代后代的分离选择重组植物。通过 PCR、Southern blot 和序列分析证实了切除，表明 OsMADS45 的切除效率为 10%至 53%，napin 的切除效率为 12%至 36%。通过 RT-PCR 分析检测到无标记转基因水稻中转 cry1Ac 和 vip3A 的表达。这些结果表明，我们的组织特异性调控 Cre/loxP 系统可以从转基因水稻中自动切除标记基因，减轻公众对转基因作物安全性的担忧。
