Qin M, Bayley C, Stockton T, Ow D W
Plant Gene Expression Center, U.S. Department of Agriculture, Albany, CA 94710.
Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1706-10. doi: 10.1073/pnas.91.5.1706.
We report the use of the bacteriophage P1 Cre-lox system for generating conservative site-specific recombination between tobacco chromosomes. Two constructs, one containing a promoterless hygromycin-resistance gene preceded by a lox site (lox-hpt) and the other containing a cauliflower mosaic virus 35S promoter linked to a lox sequence and the cre coding region (35S-lox-cre), were introduced separately into tobacco plants. Crosses between plants harboring either construct produced plants with the two constructs situated on different chromosomes. Plants with recombination events were identified by selecting for hygromycin resistance, a phenotype expressed upon recombination. Molecular analysis showed that these recombination events occurred specifically at the lox sites and resulted in the reciprocal exchange of flanking host DNA. Progenies of these plants showed 67-100% cotransmission of the new transgenes, 35S-lox-hpt and lox-cre, consistent with the preferential cosegregation of translocated chromosomes. These results illustrate that site-specific recombination systems can be useful tools for the large-scale manipulation of eukaryotic chromosomes in vivo.
我们报道了利用噬菌体P1 Cre-lox系统在烟草染色体间产生保守的位点特异性重组。将两个构建体分别导入烟草植株，一个构建体含有一个位于潮霉素抗性基因前的lox位点（lox-hpt），该潮霉素抗性基因无启动子，另一个构建体含有与lox序列和cre编码区相连的花椰菜花叶病毒35S启动子（35S-lox-cre）。携带任一构建体的植株之间杂交产生了两个构建体位于不同染色体上的植株。通过选择潮霉素抗性来鉴定发生重组的植株，重组时会表现出这种抗性表型。分子分析表明，这些重组事件特异性地发生在lox位点，导致侧翼宿主DNA的相互交换。这些植株的后代显示新转基因35S-lox-hpt和lox-cre的共传递率为67%-100%，这与易位染色体的优先共分离一致。这些结果表明，位点特异性重组系统可成为体内大规模操纵真核染色体的有用工具。
