RESUMEN
The family members of PEX11 are key factors involved in regulation of peroxisome proliferation. Sixty-six PEX11p candidates of PEX11 gene family from 26 representative fungal species were obtained and analyzed by bioinformatic strategies. In most filamentous fungi, 2 or 3 potential PEX11ps were found, in contrast with 1 or 2 in yeast species. Compared with other fungal species, the Ascomycetes tend to have more PEX11ps, and even 5 in several individuals. The data of phylogenetic analysis and protein structure indicated that all of the PEX11ps were divided into 3 groups: I, II, and III. The members of group I and group III existed in most species, while those in group II were found only in Pezizomycotina. By MEME analysis, 5-6 conserved motifs were found in each PEX11ps. Among them,motif 8 in C-terminal had the most conservation, indicating that this motif probably plays a key role in maintaining the proper function of PEX11p.
Asunto(s)
Biología Computacional , Proteínas Fúngicas/fisiología , Peroxisomas/fisiología , Secuencia de Aminoácidos , Proliferación Celular , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Peroxinas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals (PTS1 and PTS2) in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein (GFP)-PTS1, GFP-PTS2 and red fluorescence protein (RFP)-PTS1, were constructed and introduced into Magnaporthe oryzae Guy11 cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTS1 machinery was revealed by comparing the fluorescence backgrounds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into Deltamgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement of peroxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTS1 and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Magnaporthe/genética , Magnaporthe/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , ADN de Hongos/genética , Genes Fúngicos , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mutación , Receptor de la Señal 2 de Direccionamiento al Peroxisoma , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Peroxisomas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transformación Genética , Proteína Fluorescente RojaRESUMEN
Targeted gene replacement (TGR) is an important technique for gene function analysis. With the development of genome sequencing and transformation, TGR has been applied widely to filamentous fungi, and many new systems and approaches have been established. In this paper, strategies involved in TGR in filamentous fungi were reviewed, including transformation systems, targeting vector construction, mutant selection and so on. Compared with TGR, RNAi and other techniques of gene function analysis were introduced and reviewed as well.