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内容記述 |
Postembryonic growth in land plants occurs from the meristem, a localized region that gives rise to all adult structures, such as a stem and leaves. Meristems control the continuous development of plant organs by balancing the maintenance and proliferation of undifferentiated stem cells, and directing their differentiation. Meristem establishment and maintenance is a fundamental question in plant development research.<br /> Mosses have two types of meristems: a protonema apical cell and a gametophore apical cell. The gametophore apical cell is a single meristematic cell that is maintained through self-renewal, and gives rise to such organs as the stem and leaves. In the moss Physcomitrella patens, the developmental process of the apical cell is well defined at the cellular level, and gene targeting based on homologous recombination is feasible. Thus, apical cell differentiation in P. patens is used as a model system for studies of meristem development in land plants.<br /> This study investigated apical cell differentiation of gametophores in P. patens by identifying the genes expressed during this differentiation. First, gene-trap and enhancer-trap systems were established in P. patens. These techniques are useful for cloning genes and enhancers that function in specific tissues or cells. In addition, the systems are convenient for obtaining molecular markers specific to certain developmental processes. Elements for the two systems were constructed using a uidA reporter gene with a splice acceptor in the case of the gene-trap system, and a minimal promoter for the enhancer-trap system. The homologous recombination method allowed a high rate of transformation, finding 235 gene-trap and 1073 enhancer-trap lines with variable expression patterns from a total of 5637 gene-trap and 3726 enhancer-trap transgenic lines.<br /> To assess the feasibility of isolating a trapped gene, one gene-trap line, YH209 with rhizoid-specific GUS expression, was characterized. UidA-fused fragments were amplified by the 5' RACE method using nil-specific primers. One of the amplified fragments was used to screen the mini-transposon-tagged genomic DNA library that was used to generate the P. patens gene-trap lines. A genomic fragment containing the sequence of 5' RACE fragments was obtained. This fragment was re-integrated into the P. patens genome by homologous recombination, confirming that the fragment-integrated transformants exhibited rhizoid-specific expression patterns observed in the YH2O9 line. In addition, a full-length cDNA was isolated by the 3' RACE method, and the gene was named PpGLU. PpGLU forms a clade with the acidic alpha-glucosidase genes of plants. The gene-trap and enhancer-trap systems should be useful for identifying cell-type and tissue-specific genes in P. patens.<br /> From the 235 gene-trap lines and the 1073 enhancer-trap lines, three and four lines, respectively, were isolated. The isolated lines were exhibiting GUS activity preferentially in the apical cells of buds. One gene-trap line, Apicar1, showing GUS activity predominantly in the apical cell of caulonemata, rhizoids, and gametophores, was further characterized. The candidate trapped gene was isolated by both the 5' and 3' RACE methods, using the same approach as used with the YH209 line. A sequence analysis of the isolated cDNA revealed that the trapped gene encoded a kinesin-like protein, API1 (Apicar1). According to a phylogenetic analysis of API1 and kinesin superfamily genes, the API1 gene formed a new family of kinesin-like proteins with one of the Arabidopsis kinesin-like genes. This suggests that API1 may have a novel function that is different from those of kinesins of other subfamilies. API1 will also be useful as a molecular marker in studies of the establishment and maintenance of the apical cell. |
要旨・審査要旨 / Abstract, Screening Result (257.0 kB)
