tailieunhanh - Báo cáo khoa học: Survey of the Botrytis cinerea chitin synthase multigenic family through the analysis of six euascomycetes genomes

We describe a strategy for systematic amplification of chitin synthase genes (chs) in the filamentous ascomycetes plant-pathogenBotrytis cinereausing PCR with multiple degen-erate primers designed on specific and conserved sequence motifs. Eight distinctchsgeneswere isolated, namedBcchs I, II, IIIa, IIIb, IV, V, VIandVII. They probably constitute the entire chsmultigenic family of this fungus, as revealed by careful analysis of six euascomycetes I, IIIa,IIIb,IVandVIgenes were subjected to DNA walking and their deduced amino acid sequences were compared by hydrophobic cluster analysis (HCA) to localize putative residues critical for CHS activity. . | Eur. J. Biochem. 271 2153-2164 2004 FEBS 2004 doi Survey of the Botrytis cinerea chitin synthase multigenic family through the analysis of six euascomycetes genomes Mathias Choquer1 Martine Boccara2 Isabelle R. Goncalves3 Marie-Christine Soulie2 and Anne Vidal-Cros1 1UMR 7613 CNRS Universite Paris VI 2UMR 217 INRA INAP-G Universite Paris VI 3Structure et Dynamique des Genomes Institut Jacques Monod Universites Paris VI Paris VII Paris France We describe a strategy for systematic amplification of chitin synthase genes chs in the filamentous ascomycetes plantpathogen Botrytis cinerea using PCR with multiple degenerate primers designed on specific and conserved sequence motifs. Eight distinct chs genes were isolated named Bcchs I II IIIa IIIb IV V VI and VII. They probably constitute the entire chs multigenic family of this fungus as revealed by careful analysis of six euascomycetes genomes. Bcchs I IIIa IIIb IV and VI genes were subjected to DNA walking and their deduced amino acid sequences were compared by hydrophobic cluster analysis HCA to localize putative residues critical for CHS activity. HCA also enabled us to highlight three different transmembrane topologies of the CHS membranous isoenzymes. We found that the N-ter-minal region of the BcCHSI isoenzyme and its orthologues in other euascomycetes probably contain folded peptide motifs with conserved tyrosine residues. Their putative role is discussed. The BcCHSVII isoenzyme appeared to belong to a new class of CHS orthologues that was demonstrated by phylogenetic study to branch apart from division 1 and 2 of CHS. Keywords filamentous fungus glycosyltransferase hydrophobic cluster analysis phylogeny plant pathogen. The cell wall of filamentous fungi is a protective structure mainly composed of the interconnected polysaccharides chitin b-glucan mannan and galactofuranose 1 . Chitin the homopolymer of N-acetylglucosamine GlcNAc represents only 10-20 of the cell wall .

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