CSIRO Plant Industry, Canberra, ACT 2601, Australia; New South Wales Agricultural Genomics Centre, Wagga Wagga, NSW 2678, Australia.
Plant Sci. 2006 Sep;171(3):308-22. doi: 10.1016/j.plantsci.2006.03.019. Epub 2006 Apr 24.
To gain further understanding of the mechanisms involved in Agrobacterium-mediated genetic transformation and T-DNA integration, we analysed 156 T-DNA/rice, 69 T-DNA/T-DNA and 11 T-DNA/vector backbone (VB) junctions, which included 171 left borders (LB) and 134 right borders (RB). Conserved cleavage was observed in 6% of the LB and 43% of the RB. Terminal microhomology (1-10bp) was identified in 58% of T-DNA/rice, 43% of T-DNA/T-DNA and 82% of T-DNA/VB junctions, and this occurred particularly at the LB junctions. Approximately 32% of both T-DNA/rice and T-DNA/T-DNA junctions harboured 1-344bp of filler DNA that was derived mainly from the T-DNA region adjacent to the breakpoint and/or from the rice genome flanking the T-DNA integration site. Structure of the filler DNA was more complicated at the T-DNA/T-DNA junction than at the T-DNA/rice junction, indicating the presence of T-DNA recombination or rearrangement prior to or during T-DNA integration. When two T-DNAs were integrated in the inverted repeat configuration, significant truncation was always observed in one of the two T-DNAs whereas with direct repeat configuration, a large truncation was less frequent. Most integration events analysed in this study could be addressed by previously proposed models; however, the characteristics of the T-DNA repeats and the complicated filler DNA between two T-DNA copies imply that multiple mechanisms are involved in the formation of T-DNA repeats as well as in T-DNA integration in plants.
为了进一步了解农杆菌介导的遗传转化和 T-DNA 整合的机制，我们分析了 156 个 T-DNA/水稻、69 个 T-DNA/T-DNA 和 11 个 T-DNA/载体骨架（VB）接头，其中包括 171 个左边界（LB）和 134 个右边界（RB）。在 6%的 LB 和 43%的 RB 中观察到保守切割。在 58%的 T-DNA/水稻、43%的 T-DNA/T-DNA 和 82%的 T-DNA/VB 接头中鉴定出末端微同源性（1-10bp），并且这种情况特别发生在 LB 接头处。大约 32%的 T-DNA/水稻和 T-DNA/T-DNA 接头都含有 1-344bp 的填充 DNA，这些填充 DNA 主要来自与断点相邻的 T-DNA 区域和/或来自 T-DNA 整合位点侧翼的水稻基因组。填充 DNA 的结构在 T-DNA/T-DNA 接头处比在 T-DNA/水稻接头处更为复杂，这表明在 T-DNA 整合之前或期间存在 T-DNA 重组或重排。当两个 T-DNA 以反向重复构型整合时，在两个 T-DNA 中的一个中总是观察到显著的截断，而在直接重复构型中，大的截断则不太常见。本研究中分析的大多数整合事件都可以用以前提出的模型来解释；然而，T-DNA 重复的特征和两个 T-DNA 拷贝之间复杂的填充 DNA 表明，多个机制参与了 T-DNA 重复的形成以及植物中 T-DNA 的整合。
