Stuurman J, de Vroomen M J, Nijkamp H J, van Haaren M J
Department of Genetics, Free University Amsterdam, Netherlands.
Plant Mol Biol. 1996 Dec;32(5):901-13. doi: 10.1007/BF00020487.
With the aim of developing new techniques for physical and functional genome analysis, we have introduced the Cre-lox site-specific recombination system into the cultivated tomato (Lycopersicon esculentum). Local transposition of a Ds(lox) transposable element from a T-DNA(lox) on the long arm of chromosome 6 was used to position pairs of lox sites on different closely linked loci. In vitro Cre-lox recombination between chromosomal lox sites and synthetic lox oligonucleotides cleaved the 750 Mb tomato genome with 34 bp specificity to release unique 65 kb and 130 kb fragments of chromosome 6. Parallel in vitro experiments on Saccharomyces cerevisiae chromosomes show the efficiency of cleavage to be 50% per chromosomal lox site at maximum. By expressing the Cre recombinase in tomato under control of a constitutive CaMV 35S promoter, efficient and specific somatic and germinal in planta inversion of the 130 kb fragment is demonstrated. The combined use of in vitro and in vivo recombination on genetically mapped lox sites will provide new possibilities for long range restriction mapping and in vivo manipulation of selected tomato genome segments.
为了开发物理和功能基因组分析的新技术，我们已将Cre-lox位点特异性重组系统引入栽培番茄（Lycopersicon esculentum）。利用Ds(lox)转座元件从6号染色体长臂上的T-DNA(lox)进行局部转座，将lox位点对定位在不同的紧密连锁位点上。染色体lox位点与合成lox寡核苷酸之间的体外Cre-lox重组以34 bp的特异性切割了750 Mb的番茄基因组，释放出6号染色体独特的65 kb和130 kb片段。在酿酒酵母染色体上进行的平行体外实验表明，每个染色体lox位点的最大切割效率为50%。通过在组成型CaMV 35S启动子的控制下在番茄中表达Cre重组酶，证明了130 kb片段在植物体内高效且特异性的体细胞和生殖细胞内倒位。在遗传定位的lox位点上结合使用体外和体内重组，将为长距离限制图谱绘制和选定番茄基因组片段的体内操作提供新的可能性。
