Amino acid substitution in α-helix 7 of Cry1Ac δ-endotoxin of Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa armigera Hubner

AbstractInsecticidal proteins or δ-endotoxins of Bacillus thuringiensis are highly toxic to a wide range of agronomically important pests. The toxins are formed of three structural domains. The N-terminal domain is a bundle of eight α-helices and is implicated in pore formation in insect midgut epithelial membranes. All the δ-endotoxins share a common hydrophobic motif of eight amino acids in α-helix 7. A similar motif is also present in fragment B of diphtheria toxin (DT). Site-directed mutagenesis of Cry1Ac δ-endotoxin of B. thuringiensis was carried out to substitute its hydrophobic motif with that of DT fragment B. The mutant toxin was shown to be more toxic to the larvae of Helicoverpa armigera (cotton bollworm) than the wild-type toxin. Voltage clamp analysis with planar lipid bilayers revealed that the mutant toxin opens larger ion channels and induces higher levels of conductance than the wild-type toxin

Glycine Supports in Vivo Reduction of Nitrate in Barley Leaves

Glycine, a photorespiratory intermediate, enhanced the in vivo reduction of nitrate in barley (Hordeum vulgare L.) leaf slices, when included in the assay medium. Isonicotinyl hydrazide, an inhibitor of glycine oxidation, partially reduced NO(2)(−) production. The enhancement caused by glycine treatment was reversed by isonicotinyl hydrazide when both were present together in the medium. Similar effects were observed when the excised leaves were preincubated with the metabolite and the inhibitor. Glycine also partially relieved the inhibition of nitrate reduction caused by malonate, an inhibitor of the tricarboxylic acid cycle. The results support the hypothesis that glycine decarboxylation activity is a source of NADH for nitrate reductase activity

Insect-resistant transgenic brinjal plants

A synthetic cry1Ab gene coding for an insecticidal crystal protein (ICP) of Bacillus thuringiensis (Bt) was transferred to brinjal (eggplant) by cocultivating cotyledonary explants with Agrobacterium tumefaciens. Transformant plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and mRNA expression. Double-antibody sandwich ELISA analysis revealed Bt toxin protein expression in the transgenic plants. The expression resulted in a significant insecticidal activity of transgenic brinjal fruits against the larvae of fruit borer (Leucinodes orbonalis). The results also demonstrated that a synthetic gene based on monocot codon usage can be expressed in dicotyledonous plants for insect control