Johann Heinrich von Thünen-Institut, Federal Research Institute for Rural Areas, Forestry and Fisheries, Institute of Forest Genetics, Grosshansdorf, Germany.
Plant Biol (Stuttg). 2010 Mar;12(2):334-40. doi: 10.1111/j.1438-8677.2009.00293.x.
Two site-specific recombination systems, Cre/lox and FLP/FRT, were tested for marker gene removal and targeted gene transfer in a model tree system. A hybrid aspen clone (Populus tremula x Populus tremuloides) was co-transformed with plasmids containing either the FLP or the Cre recombinase, both under control of a heat-inducible promoter (HSP, Gmhsp17.5-E from soybean) flanked by the two recognition sites (FRT or lox). Molecular investigations of heat-shock treated Cre or FLP transgenic lines indicate excision of inserts between the two recognition sites. Further, a site-specific recombination at the FRT sites leading to targeted integration of a fragment could be demonstrated for the FLP/FRT system. Transgenic aspen carrying two constructs (each with different genes between the FRT sites) revealed (i) excision of both fragments between the FRT sites, and (ii) targeted integration of the fragment from the second construct exactly at the former position of the fragment in the first construct. These results indicate the usefulness of the two site-specific recombination systems in the tree species Populus. Combining both site-specific recombination systems, a strategy is suggested for targeted transgene transfer and removal of antibiotic marker genes.
两种位点特异性重组系统 Cre/lox 和 FLP/FRT 在一个模式树种系统中用于标记基因去除和靶向基因转移的测试。将含有 FLP 或 Cre 重组酶的质粒共转化为杂种白杨克隆（Populus tremula x Populus tremuloides），这两种重组酶都受热诱导启动子（来自大豆的 Gmhsp17.5-E）的控制，两侧是两个识别位点（FRT 或 lox）。对热休克处理的 Cre 或 FLP 转基因系的分子研究表明，两个识别位点之间的插入物被切除。此外，FLP/FRT 系统可证明在 FRT 位点处发生特异性重组，导致靶向整合片段。携带两个构建体（每个构建体之间在 FRT 位点处具有不同的基因）的转基因白杨显示：（i）FRT 位点之间的两个片段都被切除；（ii）第二个构建体的片段精确地靶向整合到第一个构建体中该片段的前位置。这些结果表明这两种位点特异性重组系统在杨树树种中具有实用性。结合这两种位点特异性重组系统，提出了一种用于靶向转基因转移和去除抗生素标记基因的策略。
