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

• Published in: Journal of Asia-Pacific entomology 2014, 17(3), , pp.319-325
• Main Authors: Shaikh, Faiyaz K., Gadge, Prafull P., Shinde, Ashok A., Padul, Manohar V., Kachole, Manvendra S.
• Format: Article
• Language: English
• Subjects: Albizia lebbeck; Gel X-ray film contact print technique; Helicoverpa armigera; Insect resistance; Trypsin inhibitors; 농수해양학
• ISSN: 1226-8615; 1876-7990
• DOI: 10.1016/j.aspen.2014.02.002

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. Characterization of the AlTI13 protein from Indian siris (Albizia lebbeck) that inhibits the growth of cotton bollworm (Helicoverpa armigera). [Display omitted] •AlTI13 protein of Albizia lebbeck is characterized against H. armigera.•Multiple AlTIs reported by gel X-ray contact print technique.•AlTI13 protein with MW ~28KDa indicated the IC50 for trypsin 0.14μmol/ml and for HaGPs 0.17μmol/ml.•Growth of H. armigera was severely affected on diet containing AlTIs and AlTI13.•AlTI13 may be used as a transgene for developing transgenic plants.