Dickinson Lauren, Yuan Wenxin, LeBlanc Chantal, Thomson Geoffrey, Wang Siyuan, Jacob Yannick
Yale University, Department of Molecular, Cellular and Developmental Biology, Faculty of Arts and Sciences; New Haven, Connecticut 06511, USA.
Yale University, Department of Genetics, Yale School of Medicine; New Haven, Connecticut 06510, USA.
bioRxiv. 2023 Mar 6:2023.03.05.531200. doi: 10.1101/2023.03.05.531200.
Transformation via (Agrobacterium) is the predominant method used to introduce exogenous DNA into plants. Transfer DNA (T-DNA) originating from Agrobacterium can be integrated as a single copy or in concatenated forms in plant genomes, but the mechanisms affecting final T-DNA structure remain unknown. In this study, we demonstrate that the inclusion of retrotransposon (RT)-derived sequences in T-DNA can increase transgene copy number by more than 50-fold in (Arabidopsis). RT-mediated amplification of T-DNA results in large concatemers in the Arabidopsis genome, which are primarily induced by the long terminal repeats (LTRs) of RTs. T-DNA amplification is dependent on the activity of DNA repair proteins associated with theta-mediated end joining (TMEJ). Finally, we show that T-DNA amplification can increase the frequency of targeted mutagenesis and gene targeting. Overall, this work uncovers molecular determinants that modulate T-DNA copy number in Arabidopsis and demonstrates the utility of inducing T-DNA amplification for plant gene editing.
通过农杆菌介导的转化是将外源DNA导入植物的主要方法。源自农杆菌的转移DNA（T-DNA）可以以单拷贝形式或串联形式整合到植物基因组中，但影响最终T-DNA结构的机制仍不清楚。在本研究中，我们证明在T-DNA中包含逆转座子（RT）衍生序列可使拟南芥中的转基因拷贝数增加50倍以上。RT介导的T-DNA扩增在拟南芥基因组中产生大的串联体，这主要是由RT的长末端重复序列（LTR）诱导的。T-DNA扩增依赖于与θ介导的末端连接（TMEJ）相关的DNA修复蛋白的活性。最后，我们表明T-DNA扩增可以增加靶向诱变和基因靶向的频率。总体而言，这项工作揭示了调节拟南芥中T-DNA拷贝数的分子决定因素，并证明了诱导T-DNA扩增用于植物基因编辑的实用性。
