Gene Targeting in Plants

基因组测序项目提供了大量关于植物基因组中携带的基因的知识。 这些项目的最终目标是了解这些基因中每一个的功能。 然而，DNA序列信息通常只暗示潜在的功能，需要实验室实验来表征基因作用的细节。 表征基因功能的最有力的方法涉及突变生物体的分析。 因此，迫切需要开发可以使用DNA序列信息对植物基因组进行定向修饰的方法。 最理想的技术将提供进行多种定向修饰的能力，并且不限于功能性敲除。 一种这样的技术是通过同源重组的基因靶向，其广泛用于酵母和小鼠。 然而，一个广泛使用的方法，为高等植物基因的靶向改造还没有。本项目的目标是建立一种基于同源重组的高等植物基因打靶方法。 在该过程中，将三个构建体引入基因组中。 第一个和第二个是嵌合基因构建体，其能够调节Cre蛋白酶和I-SceI基因的表达。 第三个构建体是置于两个loxP位点之间的修饰的靶基因。 修饰的靶基因含有插入基因中间的I-SceI识别序列。 在含有所有三种构建体的植物中，诱导Cre蛋白酶和I-SceI基因的表达，这些酶催化细胞核中末端断裂的染色体外DNA分子的形成，并且染色体外DNA分子与靶基因之间的同源重组产生靶基因的两个串联拷贝。 该基因靶向程序可用于修饰靶基因或将修饰引入靶基因。 为了加快基本基因靶向程序的开发，最初将使用拟南芥作为测试系统。 它的生成时间短，尺寸小，将允许快速的技术开发和测试。 此外，它的小基因组可能会增加同源重组频率。 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，因此我们在拟南芥中开发的方法很可能在包括商业上重要的作物植物在内的各种物种中起作用。 因此，所提出的实验开发的方法应有利于未来的努力，以提高作物产量和质量。

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.