Gene targeting (GT) is a technique of modifying a target DNA on a genome at will by recombination utilizing the base sequence homology of DNAs. In the field of plants also, this technique is very promising in the fundamental research and in the development of breeding materials.
Nevertheless, the frequency of the homologous recombination in higher plants is low. When vector shaving a certain mutation on a sequence homologous to a target DNA (GT vectors) are introduced into cells from the outside to modify the target DNA via GT, most of the vectors are randomly inserted into the genomes. Against this background, positive-negative selection has been developed to efficiently select cells in which GT has successfully occurred. This method is a selection method in which cells having GT vectors randomly incorporated in the genomes are eliminated on the basis of the expression of a negative selectable marker gene, while cells having mutations introduced in target DNAs by GT are isolated on the basis of the expression of a positive selectable marker gene (NPL 1).
However, when this method is used, the expression cassette of the positive selectable marker gene remains in the target DNA. Accordingly, this cassette needs to be removed in a case where only a required mutation is to be introduced into a target DNA. In this regard, there has been a report so far on a system in which a positive selectable marker gene is removed after GT using a site-specific recombinase. Nevertheless, when this system is used, the recognition sequence of the site-specific recombinase remains after the marker is removed. Since it is also reported that even inserting a short base sequence influences the expressions of adjacent genes, there has been a demand for the development of a technique capable of marker removal without leaving any footprint after GT, and used when an introduction system is constructed for a mutation equivalent to a spontaneous mutation.
In relation to the technique of removing a marker gene, the present inventors have revealed the following findings. To be more specific, first, a T-DNA having a reporter gene in which a marker gene and recognition sites of a nuclease I-SceI disposed on both ends of the marker gene are inserted is introduced into plant cells. Then, the marker gene can be removed from the reporter gene by expressing I-SceI in a plant cell in which the reporter gene is randomly inserted in the genomic DNA. In this approach, as in the case of utilizing the above-described site-specific recombinase, if the two I-SceI recognition sites are simply utilized to excise the selectable marker gene, broken ends are rejoined, leaving the I-SceI recognition sites in the genomic DNA. For this reason, the present inventors have further devised a scheme of matching (overlapping) 600-bp sequences located outside the recognition sites in the T-DNA. Thereby, after the marker gene is excised, homologous recombination occurs between the overlapping DNA sequences of broken ends, and the I-SceI recognition sites are also successfully removed (NPL 2).
However, in the method described in NPL 2, homologous recombination occurs between the overlapping DNA sequences before I-SceI is expressed. As a result, the marker gene is removed in quite a large amount from the genomic DNAs. This brings about a problem that it is difficult to select a plant cell, in which a reporter gene is randomly inserted in the genomic DNA, based on an expression of the marker gene.
Hence, for the application to GT of such a marker gene removal technique via homologous recombination between overlapping DNA sequences, it has been required to further develop a technique for stably selecting a cell, in which a mutation is introduced in a target DNA, based on an expression of a marker gene.