Department of Plant Genetics, Institute of Field and Garden Crops, ARO, The Volcani Center, P.O. Box 6, 50250, Bet Dagan, Israel.
Theor Appl Genet. 1996 Aug;93(3):407-13. doi: 10.1007/BF00223183.
FLP/FRT-mediated site-specific recombination was studied with a recombination-reporter gene system which allows visualization of β-glucuronidase (GUS) expression after site-specific excisional activation of a silent gusA gene. This system was used for characterization of the functional activity of the Saccharomyces cerevisiae native FLP recombinase driven by the cauliflower mosaic virus (CaMV) 35s promoter [linked to the tobacco mosaic virus (TMV) omega translational leader] in mediating site-specific recombination of chromosomal FRT sites in tobacco FLP x FRT-reporter hybrids. Six hybrids were generated from crosses of lines containing either a stably integrated recombination-reporter or a FLP-expression construct. The activated gusA phenotype was specific to hybrid progenies and was not observed in either parental plants or their selfed progenies. Recombination efficiency in whole seedlings was estimated by the percent of radioactivity on a Southern blot which was incorporated into the recombined DNA product. Estimated efficiency mean values for the six crosses ranged from 5.2 to 52.0%. Histochemical analysis in hybrid plants visualized GUS activity with variable chimeric patterns and intensities. Recombination efficiency and GUS expression varied both among and within crosses, while higher recombination efficiency coincided with larger and more intense patterns of GUS activity. These data suggest that recombination is induced randomly during somatic developmental stages and that the pattern and intensity generated in a given plant are affected by factors imposing varibility not only between but also within crosses. Additionally, while recombination in a population of FLP/FRT hybrids may occur in all plants, recombination efficiency may still be low in any given plant. The activity of the native, as compared to a modified, FLP (Kilby et al. 1995) in the activation of transgenic traits in tobacco is discussed.
FLP/FRT 介导的位点特异性重组是通过一个重组报告基因系统研究的，该系统允许在沉默的 gusA 基因的位点特异性切除激活后观察β-葡萄糖醛酸酶（GUS）的表达。该系统用于表征由花椰菜花叶病毒（CaMV）35s 启动子[与烟草花叶病毒（TMV）ω翻译前导序列连接]驱动的酿酒酵母天然 FLP 重组酶在介导烟草 FLP x FRT-报告杂交体中染色体 FRT 位点的位点特异性重组中的功能活性。从含有稳定整合的重组报告或 FLP 表达构建体的品系杂交产生了 6 个杂种。激活的 gusA 表型是杂种后代特有的，在亲本植物或其自交后代中均未观察到。通过 Southern 印迹上整合到重组 DNA 产物中的放射性百分比来估计整个幼苗中的重组效率。六个杂交的估计效率平均值范围为 5.2%至 52.0%。在杂种植物中的组织化学分析以可变的嵌合模式和强度可视化 GUS 活性。重组效率和 GUS 表达在杂交种之间和内部都有所不同，而较高的重组效率与更大和更强的 GUS 活性模式相关。这些数据表明，重组在体细胞发育阶段随机诱导，并且在给定植物中生成的模式和强度不仅受到杂交种之间而且受到杂交种内部的可变性因素的影响。此外，尽管在 FLP/FRT 杂种群体中可能会发生重组，但在任何给定植物中，重组效率可能仍然很低。讨论了与改良的（Kilby 等人，1995）FLP 相比，在烟草中转基因性状激活中的天然，FLP 的活性。
