A Homologous Recombination System for Plants Based on Zinc Finger Nucleases

植物具有非凡的生物合成能力，可以用来生产大量复杂分子。下一代作物将合成对食品、医药和工业有价值的特殊化学品和蛋白质。然而，要充分利用植物的生物合成潜力，就需要了解植物控制生长发育和对环境信号和胁迫作出反应的遗传基础。一旦了解了植物基因的功能，就有可能修改植物基因组，创造出满足对植物衍生产品日益增长的需求的作物。识别植物基因功能和重新编排植物遗传密码需要复杂的工具来操纵植物基因组。具体来说，需要对植物遗传密码进行各种改变，包括精确的DNA插入、删除或替换。基因靶向或同源重组是一种允许将特定DNA序列改变引入植物染色体的方法。从根本上说，基因靶向是一种DNA交换反应。将携带所需序列的DNA片段引入植物细胞，取代原有的基因拷贝。为了提高基因靶向的效率，在修饰位点（靶标）产生染色体断裂。一种叫做锌指核酸酶（ZFN）的酶被用来产生染色体断裂。ZFNs有两个组成部分：一个DNA识别域（锌指阵列）和一个切割染色体的核酸酶。锌指阵列可以识别植物基因组中的任何位点，从而使其可以修饰任何染色体序列。目前的研究方向是开发广泛应用的锌指核酸酶辅助基因靶向，包括建立高频基因靶向的关键参数和设计锌指阵列的稳健方法。该研究项目的成果将是一个高度便捷的基因靶向系统，可用于各种植物物种的基因功能研究。基因靶向平台还将使具有新特性的作物作物工程成为可能，包括那些更好地抵御害虫、具有更高的食品价值和生产具有工业重要性的化合物的作物。由于基因靶向以高度特异性和可控的方式引入植物基因组的变化，通过基因靶向产生的作物可能比传统的转基因作物更受公众接受。这可能会减少对转基因作物施加的壁垒，从而增加美国商品在全球市场上的价值。

Plants have remarkable biosynthetic capacities that can be harnessed to produce a vast array of complex molecules. The next generation of crops will synthesize specialty chemicals and proteins of value for food, medicine and industry. Fully harnessing the biosynthetic potential of plants, however, requires understanding the genetic basis by which plants control growth and development and respond to environmental signals and stresses. Once an understanding of plant gene function is attained, it will be possible to modify plant genomes to create crops that meet the burgeoning need for plant-derived products. Discerning plant gene function and re-orchestrating the plant genetic code require sophisticated tools to manipulate plant genomes. Specifically, it would be desirable to make a variety of alterations to the plant genetic code, including precise DNA insertions, deletions or substitutions. Gene targeting or homologous recombination is a method that allows specific DNA sequence changes to be introduced into plant chromosomes. Fundamentally, gene targeting is a DNA swapping reaction. A DNA fragment carrying a desired sequence is introduced into a plant cell, and it replaces the native copy of the gene. To enhance the efficiency of gene targeting, a chromosome break is created at the site of modification (the target). An enzyme called a zinc finger nuclease (ZFN) is used to generate the chromosome break. ZFNs have two components: a DNA recognition domain (a zinc finger array) and a nuclease that cleaves the chromosome. Zinc finger arrays can be designed to recognize any site in the plant genome, thereby making it possible modify any chromosomal sequence. Current research is directed at developing zinc finger nuclease-assisted gene targeting for widespread use, including establishing key parameters for high frequency gene targeting and robust methods for the design of zinc finger arrays. The outcome of this research project will be a highly facile gene targeting system that can be employed in a variety of plant species to study gene function. The gene targeting platform will also enable the engineering of crop plants with novel traits, including those that better withstand pests, have enhanced food value, and produce compounds of industrial importance. Because gene targeting introduces changes in plant genomes in a highly specific and controlled manner, crops generated through gene targeting may be met with greater public acceptance than traditional genetically modified crops. This may reduce barriers imposed on genetically modified crops and thereby increase the value of U.S. commodities in a global marketplace.