SGER: Homology-Based Gene Targeting in Arabidopsis

通过突变基因解剖生化功能和发育途径是功能基因组学的核心。基因可以通过核苷酸序列的改变、插入或删除而发生突变。利用T-DNA和转座子插入诱变已成为应用最广泛的植物基因突变方法。相比之下，同源依赖基因靶向，一种允许在编码或调控序列中进行精确改变的强大技术，尚未成为植物中的有用技术。已经确定了许多因素，这些因素可能导致缺乏基因靶向成功，或者可以优化以提高频率并增加检测植物中基于同源的重组事件的便利性。这些信息已被用于设计一种新的植物基因靶向策略。该项目采用了高风险策略，但如果成功，将导致一个可行的植物同源基因靶向系统的快速发展，这将在植物研究中具有非常高的回报。植物基因靶向策略旨在1)通过检测基因转换和重组来提高识别基于同源相互作用的可能性；2)减少与裸DNA和T-DNA转化相关的非法重组；3)利用双链DNA断裂刺激植物重组的观察结果；4)富集从染色体上释放重组供体分子的植物。因为已经获得的证据表明，目前策略的所有子组件都在工厂中起作用，所以将整个系统组合在一起并对其进行测试是下一步。这些实验结果将为后续研究植物重组的分子机制和确定基因靶向的有利遗传背景提供初步数据。

Dissection of biochemical functions and developmental pathways by mutating genes is central to functional genomics. Genes can be mutated by nucleotide sequence changes, insertions or deletions. Insertional mutagenesis with T-DNA and transposons has become the most widely used method for mutating plant genes. By contrast, homology-dependent gene targeting, a powerful technique that permits precise changes to be made in either coding or regulatory sequences, has not yet become a useful technique in plants. A number of factors have been identified that may contribute to the lack of gene targeting success or which can be optimized to enhance the frequency and increase the ease of detecting homology-based recombinational events in plants. This information has been used to design a new gene targeting strategy for plants. This project utilizes a high-risk strategy, however if successful, it will lead to the rapid development of a practicable system for homologous gene targeting in plants, which will have a very high pay-off in plant research. The strategy for plant gene targeting seeks to 1) enhance the probability of identifying homology-based interactions by detecting both gene conversion and recombination, 2) minimize illegitimate recombination associated with both naked DNA and T-DNA transformation, 3) make use of the observation that double-stranded DNA breaks stimulate recombination in plants and 4) enrich for plants in which the recombination donor molecule has been released from the chromosome. Because evidence has already been obtained indicating that all of the sub-components of the present strategy work in plants, putting the whole system together and testing it is the next step. The results of these experiments will provide the preliminary data for subsequent research to study molecular mechanisms in plant recombination and to identify favorable genetic backgrounds for gene targeting.