Hu Yufei, Chen Zhiyu, Zhuang Chuxiong, Huang Jilei
College of Life Sciences, South China Agricultural University, Guangzhou, 510642, China.
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou, 510642, China.
Plant J. 2017 Jun;90(5):954-965. doi: 10.1111/tpj.13523. Epub 2017 Apr 6.
Transferred DNA (T-DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double-stranded break repair (DSBR) pathway is a major model for T-DNA integration. From this model, we expect that two ends of a T-DNA molecule would invade into a single DNA double-stranded break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T-DNA integration, which has never been observed. In this work, we demonstrated it in an Arabidopsis T-DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T-DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T-DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break-junction pattern in seb19 is consistent with the result of heterogeneous T-DNA integration but not of recombination between two T-DNA insertions. We demonstrated that, in seb19, heterogeneous T-DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosomes 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T-DNA integration supports the DSBR model and suggests that two ends of a T-DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities.
来自根癌农杆菌的转移DNA（T-DNA）可以整合到植物基因组中。双链断裂修复（DSBR）途径是T-DNA整合的主要模型。根据这个模型，我们预期T-DNA分子的两端会侵入植物基因组中的单个DNA双链断裂（DSB）或独立的DSB。我们将后一种现象称为异源T-DNA整合，这从未被观察到。在这项工作中，我们在拟南芥T-DNA插入突变体seb19中证明了这一点。为了在核苷酸和染色体水平上解析由T-DNA整合引起的染色体结构变化，我们进行了反向PCR、基因组重测序、荧光原位杂交和连锁分析。我们在seb19中发现，单个T-DNA连接了两个不同的染色体位点，并导致了复杂的染色体重排。seb19中特定的断裂连接模式与异源T-DNA整合的结果一致，而不是两个T-DNA插入之间的重组结果。我们证明，在seb19中，异源T-DNA整合引发了4号和5号染色体上七个DSB的一系列错误修复，进而产生了易位、倒位、重复和缺失。异源T-DNA整合支持DSBR模型，并表明T-DNA分子的两端可以独立地整合到植物基因组中。我们的结果还显示了染色体异常的一个新来源。
