Molecular cloning and insecticidal effect of Inga laurina trypsin inhibitor on Diatraea saccharalis and Heliothis virescens

• Published in: Comparative biochemistry and physiology. Toxicology & pharmacology 2012-11, Vol.156 (3-4), p.148-158
• Main Authors: Ramos, Vanessa da S., Cabrera, Odalys G., Camargo, Eduardo L.O., Ambrósio, Alinne B., Vidal, Ramon O., da Silva, Desireé S., Guimarães, Lays C., Marangoni, Sérgio, Parra, José R.P., Pereira, Gonçalo A.G., Macedo, Maria L.R.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological control; Cloning; Cloning, Molecular; Enzyme Activation; Enzyme Assays - methods; Escherichia coli - genetics; Fabaceae - enzymology; Fabaceae - genetics; Feces - chemistry; Genes, Plant; Insecticides - pharmacology; Larva - drug effects; Larva - growth & development; Lepidoptera - drug effects; Pest Control, Biological - methods; Plant Proteins - genetics; Plant Proteins - pharmacology; Plant–insect interaction; Proteinase trypsin inhibitor; Proteolysis; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - pharmacology; Seeds - enzymology; Trypsin Inhibitors - genetics; Trypsin Inhibitors - pharmacology; Weight Loss
• ISSN: 1532-0456; 1878-1659
• DOI: 10.1016/j.cbpc.2012.07.007

Native Inga laurina (Fabaceae) trypsin inhibitor (ILTI) was tested for anti-insect activity against Diatraea saccharalis and Heliothis virescens larvae. The addition of 0.1% ILTI to the diet of D. saccharalis did not alter larval survival but decreased larval weight by 51%. The H. virescens larvae that were fed a diet containing 0.5% ILTI showed an 84% decrease in weight. ILTI was not digested by the midgut proteinases of either species of larvae. The trypsin levels were reduced by 55.3% in the feces of D. saccharalis and increased by 24.1% in the feces of H. virescens. The trypsin activity in both species fed with ILTI was sensitive to the inhibitor, suggesting that no novel proteinase resistant to ILTI was induced. Additionally, ILTI exhibited inhibitory activity against the proteinases present in the larval midgut of different species of Lepidoptera. The organization of the ilti gene was elucidated by analyzing its corresponding genomic sequence. The recombinant ILTI protein (reILTI) was expressed and purified, and its efficacy was evaluated. Both native ILTI and reILTI exhibited a similar strong inhibitory effect on bovine trypsin activity. These results suggest that ILTI presents insecticidal properties against both insects and may thus be a useful tool in the genetic engineering of plants.