SGER: Homology-Based Gene Targeting in Arabidopsis

通过突变基因来剖析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、插入或缺失而发生突变。利用T-DNA和转座子进行插入诱变已成为目前应用最广泛的植物基因突变方法。相比之下，依赖同源性的基因打靶技术是一种强大的技术，可以在编码或调控序列中进行精确的改变，但尚未成为植物中的有用技术。已经确定了许多因素，这些因素可能导致基因打靶缺乏成功，或者可以优化这些因素来提高植物中基于同源的重组事件的检测频率和简便性。这些信息已经被用来设计一种新的植物基因靶向策略。该项目采用了高风险的策略，但如果成功，它将导致在植物中快速开发实用的同源基因打靶系统，这将在植物研究中获得非常高的回报。植物基因打靶的策略试图1)通过检测基因转换和重组来提高识别基于同源的相互作用的可能性，2)减少与裸露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，以及4)丰富其中重组供体分子已从染色体中释放的植物。由于已经获得的证据表明，目前战略的所有子组成部分都在工厂中发挥作用，因此下一步是将整个系统组合在一起并进行测试。这些实验结果将为后续的研究提供初步数据，以研究植物重组的分子机制，并为基因打靶寻找有利的遗传背景。

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.