Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds |
| |
Authors: | Games Patrícia D Dos Santos Izabela S Mello Erica O Diz Mariângela S S Carvalho André O de Souza-Filho Gonçalo A Da Cunha Maura Vasconcelos Ilka M Ferreira Beatriz Dos S Gomes Valdirene M |
| |
Institution: | Patrícia D. Games, Izabela S. dos Santos, Érica O. Mello, Mariângela S.S. Diz, André O. Carvalho, Gonçalo A. de Souza-Filho, Maura Da Cunha, Ilka M. Vasconcelos, Beatriz dos S. Ferreira,Valdirene M. Gomes, |
| |
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 20 mM 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 6 kDa. 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 314 bp 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. |
| |
Keywords: | Antimicrobial peptides Plant defensin Pathogenic yeasts Phytopathogenic fungi Molecular cloning Phylogenetic analysis |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|