RESUMO
AIMS: To demonstrate the ability of Bacillus thuringiensis to penetrate as spore-crystal complex to the internal tissues of bean plants, and keep its insecticidal activity. To test the vertical transmission of the spore-crystal complex in Arabidopsis thaliana. METHODS AND RESULTS: The experimental strain was transformed with the pMUTIN-gfp plasmid which labelled the spores of B. thuringiensis HD-73 with the GFP protein. Once the rhizosphere of the bean plants was inoculated with the labelled strain, the bacterium was recovered from leaves, stems, and petioles. Furthermore, toxicity of treated plants was significantly higher than control plants when bio-assayed on cabbage looper larvae. The labelled strain was recovered from the dead insects. When the rhizosphere of A. thaliana plants was inoculated with the labelled strain, mature seeds from these plants were surface-sterilized and grown under in vitro conditions. The labelled strain was recovered from the seedlings. CONCLUSIONS: We showed that B. thuringiensis subsp. kurstaki (HD-73) in the rhizosphere can translocate to upper tissues of bean plants, and keep its insecticidal activity. Transmission of the labelled B. thuringiensis strain passed to the next generation of A. thaliana. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of B. thuringiensis as a potential facultative endophyte bacterium and the possible biotechnological repercussions are discussed.
Assuntos
Arabidopsis/microbiologia , Bacillus thuringiensis/fisiologia , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Inseticidas , Phaseolus/microbiologia , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/isolamento & purificação , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Endotoxinas/análise , Endotoxinas/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas Hemolisinas/análise , Proteínas Hemolisinas/genética , Inseticidas/análise , Larva/microbiologia , Mariposas/crescimento & desenvolvimento , Mariposas/microbiologia , RizosferaRESUMO
A combination of ion-exchange chromatography, preparative electrophoresis and gel filtration chromatography allowed a 1209-fold purification of one of the two major digestive alpha-amylases from larvae of the larger grain borer, Prostephanus truncatus Horn. The purified enzyme showed a molecular mass of 60.2 kDa, an isoelectric point of 4.7 and an optimal pH for activity of 6.0. The enzyme was heat labile and it was recognized by proteinaceous inhibitors from amaranth seeds (Amaranthus hypochondriacus), whereas extracts from maize (Zea mays) and tepary bean (Phaseolus acutifolius) produced very low inhibition. When the enzyme was measured at different stages of development, maximal activity was found in the second instar larvae. Activity drastically decreased to a very low level during the pupae stage and increased again at the adult stage. A zymogram of the different developmental stages showed two main bands of alpha-amylase activity, which almost disappeared at the pupae stage to increase again during the adult stage, revealing a new, smaller band. This new band may be required for a better adaptation of the adult insect to its new environment.
Assuntos
Amilases/metabolismo , Cornos/enzimologia , Mariposas/enzimologia , Animais , Proteínas de Insetos/metabolismoRESUMO
A digestive proteinase was isolated from larval extracts of Tribolium castaneum. The enzyme was partially purified using gel-filtration and ion-exchange chromatography. It is an acidic proteinase with a maximal activity at pH 3. Considering its inhibition by Pepstatin A, plus its selectivity to hydrolyze hemoglobin but not bovine serum albumin, it was classified as Cathepsin D proteinase. Its relative molecular weight is 22 kDa and it shows a high sensitivity to temperature. Unlike other cathepsin D found in animals, this enzyme is free of carbohydrate, and its activity is not affected by the presence of different anions which are known to affect the activity of plant aspartic proteinases.