Department of Experimental Plant Biology, Charles University, Faculty of Science, Viničná 5, 128 44, Prague 2, Czech Republic.
BMC Plant Biol. 2018 Oct 22;18(1):252. doi: 10.1186/s12870-018-1482-3.
Plant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome. When integrated into an active gene region, promoterless reporter genes placed near the T-DNA border sequence are frequently transcribed and even translated to reporter proteins, which is the principle of promoter- and gene-trap lines.
Here we show that even internal promotorless regions of T-DNAs are often transcribed. Such spontaneous transcription was observed in the majority of independently transformed tobacco BY-2 lines (over 65%) and it could effectively induce silencing if an inverted repeat was present within the T-DNA. We documented that the transcription often occurred in both directions. It was not directly connected with any regulatory elements present within the T-DNAs and at least some of the transcripts were initiated outside of the T-DNA. The likeliness of this read-through transcription seemed to increase in lines with higher T-DNA copy number. Splicing and presence of a polyA tail in the transcripts indicated involvement of Pol II, but surprisingly, the transcription was able to run across two transcription terminators present within the T-DNA. Such pervasive transcription was observed with three different T-DNAs in BY-2 cells and with lower frequency was also detected in Arabidopsis thaliana.
Our results demonstrate unexpected pervasive read-through transcription of T-DNAs. We hypothesize that it was connected with a specific chromatin state of newly integrated DNA, possibly affected by the adjacent genomic region. Although this phenomenon can be easily overlooked, it can have significant consequences when working with highly sensitive systems like RNAi induction using an inverted repeat construct, so it should be generally considered when interpreting results obtained with the transgenic technology.
农杆菌介导的植物转化的特点是将通常数量较少的 T-DNA 随机整合到基因组的不同位置。当整合到一个活跃的基因区域时，靠近 T-DNA 边界序列的无启动子报告基因通常会被转录，甚至翻译成报告蛋白，这就是启动子和基因捕获线的原理。
我们在这里表明，即使 T-DNA 的内部无启动子区域也经常被转录。这种自发转录在大多数独立转化的烟草 BY-2 系中（超过 65%）都有观察到，如果 T-DNA 内存在反向重复序列，它可以有效地诱导沉默。我们记录到转录通常发生在两个方向。它与 T-DNAs 内存在的任何调节元件没有直接联系，并且至少一些转录物是在 T-DNA 之外起始的。这种通读转录的可能性似乎在 T-DNA 拷贝数较高的系中增加。转录物的剪接和 polyA 尾的存在表明 Pol II 的参与，但令人惊讶的是，转录能够跨越 T-DNA 内存在的两个转录终止子。这种普遍的转录在 BY-2 细胞中的三个不同的 T-DNA 中被观察到，在拟南芥中也以较低的频率被检测到。
我们的结果表明，T-DNA 会发生意想不到的普遍通读转录。我们假设它与新整合 DNA 的特定染色质状态有关，可能受到相邻基因组区域的影响。尽管这种现象很容易被忽视，但当使用高度敏感的系统（如使用反向重复构建体诱导 RNAi）时，它可能会产生重大影响，因此在解释使用转基因技术获得的结果时，应该普遍考虑到这一点。
