RESUMO
Repeated gene targeting by recombinase-mediated cassette exchange (RMCE) is an efficient tool for the study of gene function and regulation because of the high predictability and repeatability of gene expression. We have developed the site-directed integration (SDI) vector system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined chromosomal locus in the absence of any coexpressed selection marker gene (Nanto et al. Plant Biotechnol J 3:203-214, 2005; Nanto and Ebinuma Transgenic Res 17:337-344, 2008; Nanto et al. Plant Cell Rep 28:777-785, 2009; Ebinuma and Nanto (2009) Marker-free targeted transformation, in Molecular techniques in crop improvement (2nd Edition). (Jain, S. M. and Brar, D. S. eds.), Springer Netherlands, pp. 527-543; Ebinuma and Nanto in preparation). The SDI vector system consists of a target vector to introduce the target cassette and an exchange vector to reintroduce the exchange cassette for gene replacement. We describe the molecular design and experimental protocol that can efficiently enrich RMCE events through the removal of randomly integrated copies and select clean marker-free targeted transgenic plants by using a negative marker.
Assuntos
Citosina Desaminase/genética , Marcação de Genes/métodos , Marcadores Genéticos , Nicotiana/genética , Recombinases/metabolismo , Agrobacterium/genética , Técnicas de Transferência de Genes , Genes de Plantas , Vetores Genéticos , Plantas Geneticamente Modificadas , Plasmídeos/genética , Recombinases/genética , Recombinação GenéticaRESUMO
The 26S proteasome is known to play pivotal roles in cell-cycle progression in various eukaryotic cells; however, little is known about its role in higher plants. Here we report that the subcellular distribution of the 26S proteasome is dynamically changed in a cell-cycle dependent manner in tobacco BY-2 cells as determined by immunostaining with anti-Rpn10 (a regulatory PA700 subunit) and anti-20S catalytic proteasome antibodies. The 26S proteasome was found to localize not only in nuclear envelopes and mitotic spindles but also in preprophase bands (PPBs) and phragmoplasts appearing in G(2) and M phases, respectively. MG132, a proteasome inhibitor, exclusively caused cell-cycle arrest not only at the metaphase but also the early stage of PPB formation at the G(2) phase and the collapse of the phragmoplast, which seems to be closely related to proteasome distribution in the cells.
Assuntos
Ciclo Celular/fisiologia , Nicotiana/fisiologia , Peptídeo Hidrolases/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Transporte/metabolismo , Ciclo Celular/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Fase G2/fisiologia , Leupeptinas/farmacologia , Metáfase/fisiologia , Microscopia Confocal , Microscopia de Fluorescência , Microtúbulos/metabolismo , Mitose/fisiologia , Complexos Multienzimáticos/metabolismo , Membrana Nuclear/metabolismo , Peptídeo Hidrolases/efeitos dos fármacos , Prófase/fisiologia , Complexo de Endopeptidases do Proteassoma , Proteínas/metabolismo , Spinacia oleracea/citologia , Fuso Acromático/metabolismo , Nicotiana/citologia , Nicotiana/efeitos dos fármacosRESUMO
The 26S proteasome is known to play central roles in the growth of many eukaryotes. However, little is known regarding its distribution in higher plants. Here, we report the spatial distribution pattern of Rpn3 (a regulatory PA700 subunit) and C2 (a subunit of the 20S proteasome) in rice ( Oryza sativa L.) seedlings as determined by in situ hybridization. The transcripts were abundantly co-expressed in the apical and marginal meristems of shoots and roots. Interestingly, these transcripts also accumulated in the leaf and ligule primordia of the shoot apex. Our results suggest that the 26S proteasome is spatially distributed among various tissues and may be involved not only in cell division but also in organ formation in higher plants.
Assuntos
Meristema/genética , Oryza/genética , Peptídeo Hidrolases/genética , Raízes de Plantas/genética , Complexo de Endopeptidases do Proteassoma , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Southern Blotting , Proteínas de Ciclo Celular/genética , Clonagem Molecular , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas , Hibridização In Situ , Dados de Sequência Molecular , Subunidades Proteicas , Homologia de Sequência de AminoácidosRESUMO
Somatic embryogenesis in cultured plant cells is an ideal system for investigating the whole process of differentiation and development from single cells to whole plants, and especially the molecular mechanism of expression of totipotency. This review reports recent progress the studies on somatic embryogenesis.
