2010: Targeted Mutagenesis in Arabidopsis Using Zinc Finger Nucleases

植物生物学的下一个挑战是辨别通过各种基因组测序项目揭示的许多基因的功能。了解基因功能的一个重要途径是研究特定基因功能被移除或敲除的后果。在模式植物拟南芥中，有许多有效的方法可以敲除许多感兴趣的基因。然而，功能分析经常因为遗传冗余或相同或密切相关基因的多个副本的存在而受挫。在植物中特别普遍的是串联基因复制，多达15%的拟南芥基因是以串联阵列组织的。将开发一种有针对性的突变方案，以增强现有的了解拟南芥基因功能的方法，特别是对于那些因遗传冗余而功能分析混乱的基因。该方法使用锌指核酸酶(ZFN)-由融合到Fokl内切酶DNA裂解结构域的锌指阵列组成的嵌合蛋白。高亲和力和高特异性的锌指阵列可以被设计成识别几乎任何目标基因。当ZFN切割靶DNA时，断裂的末端被低效修复，导致位点特异性突变。在最初的实验中，一个模型锌指核酸酶和一个包含同源靶点的报告基因将被用来优化有效恢复可遗传ZFN诱导的突变的方法。下一步，ZFN将被改造成可识别的天然拟南芥基因座，包括那些在突变时提供可观察到的表型的基因。ZFN也将被设计用于突变不会导致可观察到的表型的基因座。为了恢复这样一个基因座的突变，将采用稳健的DNA扩增和基于序列的检测方法。最后，重复基因的串联阵列将成为目标。阵列的删除将通过工程ZFN在最外层的基因内切割来完成。恢复缺失和个别突变的频率将被确定，以评估这种方法的相对效率。除了拟南芥，这种诱变方法对水稻和玉米等植物物种也很有价值，这些物种的基因组测序计划正在进行中，串联基因丰富。此外，产生和调节特定的染色体双链断裂的能力将有助于研究植物的染色体结构和DNA修复机制。可以访问基因敲除研究方案和结果的网站的网址是http://www.beckmancenter.umn.edu/html/2010.html.如果要充分利用植物来满足世界对燃料、饲料和工业原材料的迅速增长的需求，了解植物基因的功能是至关重要的。虽然在识别植物基因功能方面已经取得了重大进展，但现有的方法存在固有的局限性，特别是在将功能归因于多余或重复的基因方面。该项目的目标是实施一种有效的靶向突变方法来克服这些限制。这个项目将培养本科生实习生和研究生从事植物分子生物学的工作。来自代表性不足群体的学生将通过旨在促进少数群体参与科学的计划被招募到该项目中。

The next challenge in plant biology is to discern the function of the many genes revealed through the various genome sequencing projects. One important approach for understanding gene function is to study the consequence of removing or knocking out the function of a specific gene. In the model plant Arabidopsis, efficient methods are available to knock out many genes of interest. Functional analysis, however, is often frustrated by genetic redundancy or the presence of multiple copies of the same or closely related genes. Particularly prevalent in plants are tandem gene duplications, and as many as 15% of Arabidopsis genes are organized in tandem arrays. A targeted mutagenesis protocol will be developed to augment existing approaches for understanding Arabidopsis gene function, particularly for genes for which functional analysis has been confounded by genetic redundancy. The approach uses zinc finger nucleases (ZFNs) - chimeric proteins made up of a zinc finger array fused to the DNA cleavage domain of Fokl endonuclease. Zinc finger arrays of high affinity and specificity can be engineered to recognize virtually any target gene. Upon cleavage of the target DNA by the ZFN, the broken ends are repaired inefficiently, resulting in locus-specific mutations. In initial experiments, a model zinc finger nuclease and a reporter gene containing the cognate target site will be used to optimize methods for efficient recovery of heritable ZFN-induced mutations. Next, ZFNs will be engineered to recognized native Arabidopsis loci, including those that give observable phenotypes when mutated. ZFNs will also be engineered for a locus where mutatations do not result in an observable phenotype. To recover mutations at such a locus, robust DNA amplification and sequence-based detection methods will be employed. Lastly, a tandem array of duplicate genes will be targeted. Deletion of the array will be accomplished by engineering ZFNs to cleave within the outermost genes. The frequency of recovering deletions and individual mutations will be determined to assess the relative efficiency of this approach. In addition to Arabidopsis, this mutagenesis approach will also be valuable for plant species such as rice and maize, where genome sequencing projects are ongoing and tandem genes are abundant. Moreover, the ability to generate and regulate specific chromosomal double-strand breaks will facilitate studies of chromosome structure and DNA repair mechanisms in plants. The URL for the web site where protocols as well as results of gene knock-out studies can be accessed is http://www.beckmancenter.umn.edu/html/2010.html. Understanding the function of plant genes is critical if plants are to be fully harnessed to meet the world's burgeoning need for fuel, feed and industrial raw materials. Although significant progress has been made in discerning plant gene function, existing approaches have inherent limitations, particularly with respect to ascribing function to redundant or duplicated genes. The goal of this project is to implement an efficient targeted mutagenesis approach to overcome these limitations. This project will train undergraduate interns and graduate students for work in plant molecular biology. Students from underrepresented groups will be recruited to the project through programs designed to enhance minority participation in science.