Gene Targeting in Plants

基因组测序项目提供了丰富的关于植物基因组中携带基因的知识。这些项目的最终目标是了解每一个基因的功能。然而，DNA序列信息通常只暗示潜在的功能，并且需要实验室实验来表征基因作用的细节。表征基因功能最有力的方法是分析突变生物体。因此，迫切需要开发利用DNA序列信息对植物基因组进行定向修饰的方法。最理想的技术是能够进行多种定向修饰，而不局限于功能性敲除。其中一种技术是通过同源重组的基因靶向，广泛应用于酵母和小鼠。然而，目前还没有一种广泛使用的高等植物基因靶向修饰方法。本项目旨在建立一种基于同源重组的高等植物基因靶向方法。在这个过程中，三种结构被引入基因组。第一种和第二种是嵌合基因结构，能够调节Cre重组酶和I-SceI基因的表达。第三种结构是将修饰后的靶基因置于两个loxP位点之间。修饰后的靶基因中间插入一个I-SceI识别序列。在具有这三种结构的植物中，Cre重组酶和I-SceI基因的表达被诱导，这些酶催化细胞核内染色体外DNA分子断裂的形成，染色体外DNA分子与靶基因之间的同源重组产生两个串联的靶基因拷贝。这种基因靶向程序可用于使靶基因失活或将修饰引入靶基因。为了加快基本基因靶向程序的开发，拟南芥将最初用作测试系统。它的生成时间短，尺寸小，可以快速开发和测试技术。此外，它的小基因组可能增加同源重组频率。在这些实验中使用的方法提供了产生几乎无限种类的遗传变化的能力。由于进行同源重组的酶在整个进化过程中高度保守，我们在拟南芥中开发的方法很可能适用于各种各样的物种，包括重要的商业作物植物。因此，提出的实验开发的方法应该有利于未来提高作物产量和质量的努力。

Genome sequencing projects are providing a wealth of knowledge about the genes carried in plant genomes. The ultimate goal of these projects is to understand the function of each of these genes. However, DNA sequence information typically only hints at potential function, and laboratory experiments are needed to characterize the details of gene action. The most powerful approach to characterizing gene function involves the analysis of mutant organisms. Thus, there is a pressing need for the development of methods that can use DNA sequence information to make directed modifications of plant genomes. The most desirable technique would give the ability to make a variety of directed modifications, and not be limited to functional knockouts. One such technique is gene targeting via homologous recombination, which is widely used in yeast and the mouse. However, a widely usable method for targeted modification of higher plant genes is not yet available. The goal of this project is to produce a gene targeting methodology based on homologous recombination for higher plants. In this procedure, three constructs are introduced into the genome. The first and second are chimeric gene constructs that enable regulated expression of the Cre Recombinase and I-SceI genes. The third construct is a modified target gene placed between two loxP sites. The modified target gene contains an I-SceI recognition sequence inserted into the middle of the gene. In plants harboring all three constructs, expression of the Cre Recombinase and I-SceI genes is induced, these enzymes catalyze the formation of a broken-ended extrachromosomal DNA molecule in the nucleus, and homologous recombination between the extrachromosomal DNA molecule and the target gene generates two tandem copies of the target gene. This gene targeting procedure can be used to inactivate a target gene or to introduce modifications into a target gene. To expedite development of a basic gene targeting procedure, Arabidopsis will initially be used as the test system. Its short generation time and small size will allow rapid technology development and testing. Furthermore, its small genome may increase homologous recombination frequency. The method to be used in these experiments provides the ability to produce an almost unlimited variety of genetic changes. Because the enzymes that carry out homologous recombination have been highly conserved throughout evolution, the methods we develop in Arabidopsis are very likely to work in a broad variety of species including commercially important crop plants. Thus, the methodology developed by the proposed experiments should benefit future efforts to improve crop yield and quality.