Seqüência parcial de DNA, caracterização bioquímica da lectina de sementes de Canavalia grandiflora Benth. e suas relações com a filogenia de leguminosas

Abstract

The aim of the present work was to obtain a biochemical and molecular profiles of a new lectin isolated from seeds of Canavalia grandiflora Benth. ConGF was isolated by affinity chromatography in Sephadex G-50 and eluted by 0.1 M glucose. SOS-PAGE in the presence of 2-mercaptoehanol of ConGF yielded three distinct bands: a - chain and f3- and y- fragments (25, 50 e 100 kDa, respectively). They are similar with others lectins from sub tribe Diocleinae. ConGF amino acid composition exhibited high amount of aspartic acid, serine and leucine. Methionine and cysteine were not present. The lectin showed immunological identities with lectins from Canavalia species and partial with Dioclea rostrata but not with D. guianensis and Vatairea macrocarpa lectins. The ConGF lectin showed thermodynamic bindings profiles to deoxy analogs as observed with Dioclea lectins. The binding kinetic with glycoproteins exhibited a lectin preference for ofigomannosides, specially Man9. The seed flour PAGE-SOS with 2-mercaptoethanol showed a number of bands similar with the genus Dioclea but not with those from Crafylia floribunda and Cymbosema sp seeds. The lectin present in C. grandffora agglutinated strongly native rabbit erythrocytes and trypsin-treated human A erythrocytes. ConGF was inhibited by mannose, a-D-methyl-pyiranoside, 1-o-Methyl-a-D-glucopyranoside and by the giycoproteins porcine and bovine mucin and bovine lactotransferrin. The genetic studies showed by metaphysic chromosomes analysis on Canavaifa species 2n = 22, like the most Phaseoleae tribe. They are small and very homogeneous. The molecular studies were carried out with the cloning in Escherichia coil, the gene sequencing and the phyilogenetic analysis, including others leguminous iectins. The CTBA was efficient for extraction of DNA, showing a single band, in agarose gel, with 23.000 bp. The PCR amplification in genomic DNA and recombinant plasmids was effected with homologous primers of ConBr (Canavalia brasiliensis) gene, presenting only a band with 870 bp. The same DNA band was obtained in the PCR with plasmids from alkaline hydrolysis in E. coil clones, which confirms the transformation by the bacteria. The phylogenetic studies upon 70 amino acid sequences of Fabaceae lectins, clearly defined the Diocleinae subtribe and the Vicieae tribe. Furthermore, the results showed a evolutionary affinities between Gaiegeae-Vicieae-Trifolieae, Phaseoleae and Sophoreae-Genisteae groups. The evolutive study use the Ks and KA frequencies in nucleotide sequences of legume lectins, showed Kimura neutralist theory possibilities.