Makarevitch I, Svitashev S K, Somers D A
Department of Agronomy and Plant Genetics, University of Minnesota, 411 Borlaug Hall, 1991 Buford Circle, St. Paul, MN 55108, USA.
Plant Mol Biol. 2003 May;52(2):421-32. doi: 10.1023/a:1023968920830.
A substantial literature exists characterizing transgene locus structure from plants transformed via Agrobacterium and direct DNA delivery. However, there is little comprehensive sequence analysis of transgene loci available, especially from plants transformed by direct delivery methods. The goal of this study was to completely sequence transgene loci from two oat lines transformed via microprojectile bombardment that were shown to have simple transgene loci by Southern analysis. In line 3830, transformed with a single plasmid, one major and one of two minor loci were completely sequenced. Both loci exhibited rearranged delivered DNA and flanking genomic sequences. The minor locus contained only 296 bp of two non-contiguous fragments of the delivered DNA flanked by genomic (filler) DNA that did not originate from the integration target site. Predicted recognition sites for topoisomerase II and a MAR region were observed in the transgene integration target site for this non-functional minor locus. Line 11929, co-transformed with two different plasmids, had a single relatively simple transgene locus composed of truncated and rearranged sequences from both delivered DNAs. The transgene loci in both lines exhibited multiple transgene and genomic DNA rearrangements and regions of scrambling characteristic of complex transgene loci. The similar characteristics of recombined fragments and junctions in both transgenic oat lines implicate similar mechanisms of transgene integration and rearrangement regardless of the number of co-transformed plasmids and the level of transgene locus complexity.
已有大量文献描述了通过农杆菌介导和直接DNA导入法转化的植物中转基因位点的结构。然而，目前关于转基因位点的全面序列分析较少，尤其是来自通过直接导入法转化的植物。本研究的目的是对通过微粒轰击转化的两个燕麦品系的转基因位点进行全序列分析，Southern分析表明这两个品系具有简单的转基因位点。在3830品系中，用单个质粒进行转化，对一个主要位点和两个次要位点之一进行了全序列分析。两个位点均显示出导入的DNA和侧翼基因组序列发生了重排。次要位点仅包含导入DNA的两个不连续片段的296 bp，两侧为并非来自整合靶位点的基因组（填充）DNA。在这个无功能的次要位点的转基因整合靶位点中观察到了拓扑异构酶II的预测识别位点和一个MAR区域。11929品系用两种不同的质粒进行共转化，有一个相对简单的转基因位点，由来自两个导入DNA的截短和重排序列组成。两个品系中的转基因位点均表现出多个转基因和基因组DNA重排以及复杂转基因位点特有的混乱区域。两个转基因燕麦品系中重组片段和连接点的相似特征表明，无论共转化质粒的数量和转基因位点的复杂程度如何，转基因整合和重排的机制相似。
