Lossoffunction study of TFs in plants

A direct method for investigating the biological function of every gene is to assess the phenotype of loss-of-function mutants. For research in the Arabidopsis model system, various sources of insertional knockout mutants are readily available. For example, these materials can be accessed by the public at the following Web addresses: http://www.arabidopsis.org/abrc/ or http://www.brc.riken.jp/inf/en/. Loss-of-function mutants, from inser-tional mutagenesis or transposable elements, have provided a tremendous amount of information regarding the role of some genes. Despite these advance, however, the assignment of function to a large portion of genes remains unknown. If knockout mutant lines are not available, the accumulation of mRNA corresponding to genes of interest may also be selectively suppressed by antisense or RNA interference (RNAi) techniques. As an example for the suppression of a target gene using the antisense technique, please refer to Osakabe et al. (2002). For RNAi, we are currently using the vector system developed by CSIRO (Wesley et al., 2001). Information regarding the vector system can be accessed at http://www.pi.csiro.au/ tech_licensing_biol/genesilencingvectors.htm. For researchers who are using rice as their model system, another RNAi vector system has been developed by Miki and Shimamoto (2004) and can effectively provide transient suppression of gene function in rice. Detailed information about this vector system and its application can be obtained from the same reference.

A new method has been developed for loss-of-function studies (Hiratsu et al., 2003). This method, which is termed the dominant repression technique, was developed to overcome the functional compensation that usually occurs when using insertion mutant lines due to redundancy of homologous genes (Zhang, 2003). Hiratsu et al. (2003) showed that the expression of specific target genes was suppressed dominantly by transla-tional fusions between several TFs and a repression domain (RD) derived from the EAR motif of SUPERMAN, a TFIIIA-type zinc finger repressor. This dominant suppression occurred even in the presence of redundant TFs. Our group successfully applied this method to suppress the function of RD26 and AREB1 TFs (Fujita et al., 2004, 2005). Detailed information, including plasmid construction methods, can be obtained from Fujita et al. (2005). The same approaches described earlier were used to assess the phenotype of knockout or suppressed plants.

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