Investigation of insecticidal activity of rye α-amylase inhibitor gene expressed in transgenic tobacco (Nicotiana tabacum) toward cotton boll weevil (Anthonomus grandis)

Innumerable proteinaceous alpha-amylase inhibitors have been isolated and identified from different plant species. Among them, an alpha-amylase inhibitor gene with bioinsecticidal potential toward Anthonomus grandis (cotton boll weevil) was previously identified in rye seeds (Secale cereale). This cereal inhibitor was expressed in tobacco plants (Nicotiana tabacum) under control of phytohemaglutinin promoter by using Agrobacterium tumefasciens - mediated transformation. Presence of alpha BIII-rye gene and further protein expression were confirmed by PCR and Western blot analysis, respectively. Immunological assays indicated that the recombinant inhibitor was expressed in concentration range from 0.1% to 0.28% (w:w) of the total protein in tobacco seeds of R-0 plants. From 14 independent transformants, five plants with expression levels between 0.20% and 0.28% in seeds were in vitro assayed against A. grandis amylolytic enzymes causing clear inhibition. Moreover, bioassays using transgenic seed flour mixture for artificial diet produced 74% mortality in A. grandis first larval instar. These data suggest that rye inhibitor could be a promising biotechnological tool for produce transgenic cotton plants with an increased resistance to cotton boll weevil. Moreover, alpha BIII-rye gene should be considered a potential compound for a pyramiding strategy aiming to delay insect-resistance. (C) 2010 Published by Elsevier Inc.