Isolation and characterization of lectin gene from rice bean Vigna umbellata Thunb Ohwi and Ohashi

Abstract

newline The carbohydrate binding specificity of lectins and resistance to proteolytic degradation in insect mid gut make them potential insecticidal agents. In the present investigation, rice bean lectin gene (RbL) was amplified using degenerate primer. The amplified fragment was cloned in pGEMT- Easy vector. The sequencing of the cloned fragment resulted in 843bp gene sequence. The RbL gene was also cloned and sequenced using genomic DNA which also revealed composition of 843bp that confirmed intronless nature of rice bean lectin gene. For gene expression of the rice bean lectin gene, the coding sequence was ligated to pET-28a-c (+) expression vector, and transformed into E. coli BL- 21(DE3) cells. The expression of gene was induced using IPTG with 0.5mM and 1.0mM concentrations at 37oC for 4hr and 5hr. The highest expression of fusion protein (His6-RbL) was observed with 0.5mM IPTG concentration after induction for 4hr at 37oC. Using IMAC chromatography, the recombinant lectin (rRbL) of ~35 kDa m. wt. was purified to the extent of 0.26mg/ml. The comparative analysis of RbL gene and its deduced amino acid sequence revealed more than 90 per cent sequence similarity with Vigna aconitifoila and Vigna angularis lectins. The RbL gene encoded agglutinin precursor of 280 amino acid length with two putative signal peptide cleavage sites. Phylogenetic analysis showed a close relationship between RbL gene and other legume lectin genes. ExPASy ProtParam tool revealed acidic, stable, hydrophobic nature of putative RbL protein with estimated half life of 30hrs. The structural analysis revealed that putative RbL protein had predominance of and#946;-sheets forming the characteristic carbohydrate binding loops with an additional adenine binding site. Evaluation and validation of structured model revealed a good quality of structured 3D-model of RbL protein. and#946;-D- galactose, N-acetyl-D-glucosamine, lactose and adenine were predicted as putative ligands for RbL protein. The binding specificity of RbL to ligands was further confirmed by agglutination and agglutination inhibition assays with trypsinized human (A, B and O) and rabbit erythrocytes. RbL protein revealed conserved amino acid residues essential for carbohydrate binding and metal binding. The RbL protein was characterized with the role in plant defense using computational analysis. The results of the study would be helpful in designing future strategies for generating insect pest resistance in agriculturally important crops.