title: Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds.
abstract: The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301-9], with some modifications. A DEAE-Sepharose, equilibrated with 20mM Tris-HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS-Tricine gel electrophoresis with a molecular mass of approximately 6kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.
authors.affiliation: Universidade Estadual do Norte Fluminense, Laboratório de Fisiologia e Bioquímica de Microrganismos, Campos dos Goytacazes, 28013-602 RJ, Brazil.
authors.forename: Patrícia D
authors.initials: PD
authors.lastname: Games
authors.forename: Izabela S
authors.initials: IS
authors.lastname: Dos Santos
authors.forename: Erica O
authors.initials: EO
authors.lastname: Mello
authors.forename: Mariângela S S
authors.initials: MS
authors.lastname: Diz
authors.forename: André O
authors.initials: AO
authors.lastname: Carvalho
authors.forename: Gonçalo A
authors.initials: GA
authors.lastname: de Souza-Filho
authors.forename: Maura
authors.initials: M
authors.lastname: Da Cunha
authors.forename: Ilka M
authors.initials: IM
authors.lastname: Vasconcelos
authors.forename: Beatriz Dos S
authors.initials: Bdos S
authors.lastname: Ferreira
authors.forename: Valdirene M
authors.initials: VM
authors.lastname: Gomes
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000302:isolation & purification* / Q000494:pharmacology; D001483:Base Sequence; D049109:Cell Proliferation / Q000187:drug effects; D018076:DNA, Complementary / Q000302:isolation & purification / Q000378:metabolism*; D023082:Defensins / Q000302:isolation & purification / Q000494:pharmacology*; D008969:Molecular Sequence Data; D027805:Phaseolus / Q000737:chemistry*; D010802:Phylogeny; D012639:Seeds / Q000737:chemistry*; D015003:Yeasts / Q000187:drug effects
chemical_list: D000935:Antifungal Agents; D018076:DNA, Complementary; D023082:Defensins