SGER: Homology-Based Gene Targeting in Arabidopsis

SGER：拟南芥中基于同源性的基因打靶

• 批准号: 0130598
• 负责人: Nina Fedoroff
• 金额: $ 10万
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2002-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0130598&HistoricalAwards=false
• 关键词: SGER; Homology; Based; Gene; Targeting

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.

功能基因组学的核心是通过突变基因来剖析生物化学功能和发育途径。基因可以通过核苷酸序列改变、插入或缺失而突变。利用T-DNA和转座子进行插入突变已成为植物基因突变的最常用方法。相比之下，同源性依赖的基因靶向，一个强大的技术，允许精确的变化，无论是在编码或调节序列，尚未成为一个有用的技术在植物中。 已经鉴定了许多因素，这些因素可能导致基因靶向成功的缺乏，或者可以优化这些因素以提高频率并增加检测植物中基于同源性的重组事件的容易性。这些信息已被用于设计一种新的植物基因打靶策略。 该项目采用了高风险策略，但如果成功，它将导致植物同源基因打靶的实用系统的快速发展，这将在植物研究中产生非常高的回报。 植物基因靶向的策略寻求1）通过检测基因转换和重组两者来提高鉴定基于同源性的相互作用的可能性，2）使与裸DNA和T-DNA转化两者相关的非法重组最小化，3）利用双链DNA断裂刺激植物重组的观察结果，富集其中重组供体分子已从染色体释放的植物。 因为已经获得的证据表明，本战略的所有子组件在工厂中都有效，所以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研究植物重组的分子机制和筛选基因打靶的有利遗传背景提供初步数据。