RESUMEN
Plant cystatins show great potential as tools to genetically engineer resistance of crop plants against pests. Two important potential targets are the bean weevils Acanthoscelides obtectus and Zabrotes subfasciatus, which display major activities of digestive cysteine proteinases in midguts. In this study a cowpea cystatin, a cysteine proteinase inhibitor found in cowpea (Vigna unguiculata) seeds, was expressed in Escherichia coli and purified with a Ni-NTA agarose column. It strongly inhibited papain and proteinases from midguts of both A. obtectus and Z. subfasciatus bruchids, as seen by in vitro assays. When the protein was incorporated into artificial seeds at concentrations as low as 0.025%, and seeds were consumed by the bruchids larva, dramatic reductions in larval weight, and increases in insect mortality were observed. Molecular modeling studies of cowpea cystatin in complex with papain revealed that five N-terminal residues responsible for a large proportion of the hydrophobic interactions involved in the stabilization of the enzyme-inhibitor complex are absent in the partial N-terminal amino acid sequencing of soybean cystatin. We suggest that this structural difference could be the reason for the much higher effectiveness of cowpea cystatin when compared to that previously tested phytocystatin. The application of this knowledge in plant protein mutation programs aiming at enhancement of plant defenses to pests is discussed.
Asunto(s)
Cistatinas/química , Fabaceae/química , Modelos Moleculares , Proteínas de Plantas/química , Gorgojos , Agricultura/métodos , Secuencia de Aminoácidos , Animales , Cistatinas/genética , Cistatinas/metabolismo , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/genética , Inhibidores de Cisteína Proteinasa/metabolismo , Fabaceae/genética , Fabaceae/metabolismo , Datos de Secuencia Molecular , Control Biológico de Vectores/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ingeniería de Proteínas/métodos , Estructura Secundaria de ProteínaRESUMEN
A cowpea (Vigna unguiculata L. Walpers cv. Pitiúba) cystatin was analysed to determine its localization during development and germination of cowpea seeds, using western blotting with a specific antiserum. The pattern of immunoreactive proteins changed during development, with the major reactive bands present in dried seeds being mobilized after a 62-h period of imbibition. Immunohistochemical analysis revealed that cowpea cystatin is distributed in both embryonic axes and cotyledons with the highest level being present in the outer cell walls of the adaxial surface of the cotyledons.