Gene Targeting in Plants

University Of Utah, Salt Lake City UT

Investigators

Abstract

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.