Onouchi H, Nishihama R, Kudo M, Machida Y, Machida C
Department of Biology, Faculty of Science, Nagoya University, Japan.
Mol Gen Genet. 1995 Jun 25;247(6):653-60. doi: 10.1007/BF00290396.
Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic beta-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F1 and F2 progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F1 progeny and most of the F2 progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F2 individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F1 generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.
当来自鲁氏接合酵母的重组酶基因(R)在植物中表达时,拟南芥中的一段DNA片段可通过两个特定重组位点(RSs)之间的相互重组而被切除。为了监测重组事件,我们构建了几株携带隐性β-葡萄糖醛酸酶(GUS)报告基因的转基因拟南芥植株,该报告基因的设计方式使得其表达可由R基因介导的重组诱导。我们还构建了几个将R基因与花椰菜花叶病毒35S启动子相连的转基因株系。将每个携带隐性报告基因的转基因株系与每个携带R基因的株系进行杂交。通过组织化学方法检测F1和F2后代中GUS的活性,并通过Southern印迹和聚合酶链反应分析两个RSs之间的重组。在F1后代的幼苗和小植株以及大多数F2后代中,观察到了多种GUS活性模式,包括叶片中的扇形嵌合体。一小部分F2个体在整株植物中都表现出GUS活性。基于Southern印迹对DNA结构的分析,这种表达模式归因于F1代中的生殖细胞重组。这些结果表明,通过亲本转基因株系的异花授粉,R基因介导的重组可在拟南芥的体细胞和生殖细胞中诱导发生。