To analyze gene functions, methods for introducing a point mutation or introducing an insertion or deletion mutation into a gene are conventionally employed. For the introduction of a point mutation, a method for chemically treating the whole genome with a mutation-inducing reagent is common. However, according to this method, one nucleotide substitution must be searched for from among nucleotide sequences constituting hundreds of millions of genomic DNAs though a mutation can easily be introduced, and therefore identification takes much time. Accordingly, it may be said that the method is unsuitable for determining the identification of functions of tens of thousands of genes comprehensively and at high speeds.
A gene tagging method is known as a method for introducing a mutation into a gene with high efficiency and examining gene function in a short period of time. According to this method, a known gene fragment (a tag) is inserted into a genome at random and a gene function at the insertion site is disrupted. For plants, a T-DNA or a transposon is used as such a gene tag (Krysan, P. J. et al., Plant Cell, 1999. 11(12): pp. 2283-90; Speulman E. et al., Plant Cell, 1999. 11(10): pp. 1853-66). A gene fragment is randomly inserted into a genome by infection of a plant with Agrobacterium and by crossing with a plant having transposase, in the cases of a T-DNA and a transposon, respectively. Then, one or two copies of T-DNA are usually inserted per plant individual (Azpiroz-Leehan, R. et al., Trends Genet, 1997. 13(4): pp. 152-6), and in the case of a certain type of transposon, one copy thereof is inserted into a genome (Fedoroff, N. et al., Bioessays, 1995. 17(4): pp. 291-7). By preparing tens of thousands of such insertion mutation strains, a group of plant strains wherein individual gene functions are disrupted can be produced.
A plant exhibiting a mutant character of interest is isolated and thereafter the relationship between the mutant character and the gene is examined. In such case, genetic information adjacent to the insertion site can be obtained by methods such as PCR using the introduced gene fragment as a clue, and thereby gene function can be identified comprehensively and at high speeds (Krysan, P. J. et al., Plant Cell, 1999. 11(12): pp. 2283-90; Speulman, E. et al., Plant Cell, 1999. 11(10): pp. 1853-66).
As an improved form of this gene tagging method, an activation tagging method is known. This activation tagging method can bring about the transcriptional activation of a gene existing adjacent to the genome, into which the T-DNA has been inserted, by the use of a transcriptional enhancer sequence incorporated into the T-DNA. In recent years, this method has been developed as a new analytical method regarding plant gene functions (Walden, R. et al., Plant Mol Biol, 1994. 26(5): pp.1521-8). Among the features of this activation tagging method, a feature for enabling the production of a dominant mutation by a tag is considered to be the most important. In other words, it is possible to observe even a phenotype attributable to a gene mutation of a type having an overlapping function with other genes (e.g., gene group constituting a gene family). This feature has never been observed through the production of a conventional gene disrupted type of mutant.
However, there is one significant problem when such activation tagging method is used for comprehensive analysis of gene functions (analyzing gene functions existing on a genome in a group). That is, a genome region that can potentially be transcriptionally activated is extended to approximately 5 kb backward and forward from the insertion site since the enhancer sequence is used as an activator inside the tag (Weigel, D. et al., Plant Physiol, 2000. 122(4): pp. 1003-13). Since two or more genes are present on average in a genome region of 10 kb in a model plant like Arabidopsis thaliana, it is difficult to determine which gene is activated by the enhancer. Therefore, in order to specify a causative gene, these genes adjacent to the insertion site are all isolated and transformations thereof are carried out again for enforced expression, so that the reproduction of the phenotype is confirmed. Through this confirmation, it is essential to examine which gene has functioned to define the phenotype. This means that time on a yearly scale is required on average for analyses from the isolation of a plant exhibiting a trait of interest to the specification of a causative gene. Accordingly, in an attempt to use the activation tagging method for comprehensive analysis of genomic gene functions, the novelty of the obtained gene species or phenotype can be recognized. However, a significant contradiction that merits such as rapid specification of a gene and applicability to comprehensive analysis cannot be obtained, which can be found in the case of a conventional tagging method which is a gene disrupted type.
The present invention has an object to provide a next-generation activation tagging system, whereby a causative gene of a phenotype can easily be specified and gene functions can be comprehensively analyzed.