农杆菌介导的T-DNA整合与植物DNA双链断裂修复机制的关联研究

DNA double-strand break repair (DSBR) pathway is a major model for T-DNA integration mediated by Agrobacterium. This model suggests that T-DNA would be incorporated into endogenous DNA double-strand breaks (DSBs) by DNA repairing. However, the molecular mechanism of the DSBR model is not fully understood. In our previous report of heterogeneous T-DNA integration, we demonstrated that two ends of a single T-DNA could be integrated into different DSBs, and this event would spike a cascade of wrong DSB repairs. Heterogeneous T-DNA integration supports the DSBR model and suggests that the machinery of DSB repair in plants would be hijacked by Agrobacterium. By means of introducing adjacent DSBs into the Arabidopsis genome and 3D-FISH, we intend to reveal more features of heterogeneous T-DNA integration and look for evidence of Agrobacterium’s influence on the plant’s DSB repair. In addition, analysis of differentially expressed gene based on RNAseq technique will be used to unravel the mechanistic relationship between T-DNA integration and DSB repair in plants. Our work would contribute to understand the mechanism of both T-DNA integration and DSB repair in plants.

DNA双链断裂修复模型（DSBR）是解释农杆菌介导的T-DNA整合的一种主流假说。该模型认为外源T-DNA通过DNA末端修复整合到植物基因组的双链断裂点（DSB）中，但DSBR模型中参与T-DNA整合的分子机理并不清楚。我们之前在拟南芥中发现的heterogeneous T-DNA整合现象显示T-DNA的两个末端能够分别整合到不同的DSB中，并引发DSB级联的错误连接。该现象支持DSBR模型，并暗示农杆菌通过劫持植物内源的DSB修复机制为T-DNA插入提供机会。在此研究基础上，本项目将通过在拟南芥基因组中引入邻近的DSB及3D-FISH等手段探究heterogeneous T-DNA整合的规律，以明确农杆菌侵染对植物内源DSB修复的影响。此外，我们将利用表达谱分析寻找T-DNA整合与植物DSB修复机制关联的分子证据。本项目的开展对揭示T-DNA整合及植物DNA修复机制均有重要意义。

农杆菌介导的植物遗传转化在转基因作物育种以及植物基因功能研究方面均有重要应用，但T-DNA整合的机理并不清楚。主流观点认为T-DNA整合依赖于宿主植物的DNA修复机理，但T-DNA整合与DNA断裂修复关联的分子机制并未明确。本项目一方面探究T-DNA在拟南芥中的整合的规律，另一方面利用转录组寻求宿主植物参与T-DNA整合的候选基因。我们获得以下结果：1）基于定量PCR开发了一种高效鉴定T-DNA单拷贝的方法，即SC-PCR。该方法简单而准确，较southern blot适合大规模植株的筛选，为探究T-DNA整合规律提供了新的手段。2）反向串联的T-DNA结构会影响基于PCR原理开发的检测T-DNA拷贝数的准确性，限制性酶切基因组可以解决这一问题；3）单拷贝T-DNA整合在无骨架的转基因拟南芥中富集。这一发现结合SC PCR技术大大提高了寻找单拷贝转基因植株的效率。4）烟草DNA修复相关基因对农杆菌侵染存在不同程度的转录响应。转录响应与毒蛋白有关，而与T-DNA无关。这一结果表明农杆菌利用分泌的毒蛋白控制植物DNA修复机制，以便于T-DNA整合。5）利用拟南芥组成型或诱导型表达农杆菌毒蛋白VirD2，通过转录组分析寻找了参与T-DNA整合的候选基因。此方法简化了农杆菌侵染引发宿主植物产生的复杂响应，有可能找到参与T-DNA整合的相关基因。6）利用分步法CRISPR/Cas9探讨T-DNA的定点整合规律，发现分步法并不能提高编辑效率以及产生T-DNA的定点整合。