Characterization of the AlTI13 protein from Indian siris (Albizia lebbeck) that inhibits the growth of cotton bollworm (Helicoverpa armigera)

Abstract

Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Two groups of AlTIs—1 major (10 AlTIs; slow migration on the gel) and 1 minor (7 AlTIs; fast migration on the gel) were identified. The former was specific only toward trypsin. However, the latter was specific toward both trypsin and Helicoverpa armigera gut proteinases (HaGPs). The most potent AlTI (AlTI13) was purified to assess its in vivo bioefficacy toward HaGPs. Purification was achieved using (NH4)2SO4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH4)2SO4 F3 fractions (0.1%, 0.5%, and 1%) were approximately 79%, 83%, and 90%, respectively, resulting in reductions in the average larval weight of H. armigera. Artificial diet containing a single dose of AlTI13 (5μg/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitory concentrations (IC50) of AlTI13 for trypsin and HaGPs were 0.14 and 0.17μmol/ml, respectively. The optimum conditions for AlTI13 were pH8 and temperatures ranging from 35 to 40°C. Reducing sodium dodecyl sulfate-PAGE analysis indicated that ~28kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that AlTIs, particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.