Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis
ABSTRACT Twenty-four serovars of Bacillus thuringiensis (Bt) were screened by polymerase chain reaction to detect the presence of vegetative insecticidal protein gene (vip)-like sequences by using vip3Aa1-specific primers. vip-like gene sequences were identified in eight serovars. These genes were cloned and sequenced. The deduced amino acid sequence of the vip3Aa14 gene from Bacillus thuringiensis tolworthi showed considerable differences as compared to those of Vips reported so far. The vip3Aa14 gene from Bt tolwarthi was expressed in Escherichia coli using expression vector pET29a. The expressed Vip3Aa14 protein was found in cytosolic supernatant as well as pellet fraction, but the protein was more abundant in the cytosolic supernatant fraction. Both full-length and truncated (devoid of signal sequence) Vips were highly toxic to the larvae of Spodoptera litura and Plutella xylostella. Truncation of Vip3Aa14 protein at N-terminus did not affect its insecticidal activity.
Full-textDOI: · Available from: Siva K Panguluri, Dec 18, 2013
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ABSTRACT: Vip3 proteins have been described to be secreted by Bacillus thuringiensis during the vegetative growth phase and to display a broad insecticidal spectrum against lepidopteran larvae. Vip3Aa protoxin has been reported to be significantly more toxic to Spodoptera frugiperda than to Spodoptera exigua and differences in the midgut processing have been proposed to be responsible. In contrast, we have found that Vip3Ae is essentially equally toxic against these two species. Proteolysis experiments were performed to study the stability of Vip3A proteins to peptidase digestion and to see whether the differences found could explain differences in toxicity against these two Spodoptera species. It was found that activation of the protoxin form and degradation of the 62 kDa band took place at lower concentrations of trypsin when using Vip3Aa than when using Vip3Ae. The opposite effect was observed for chymotrypsin. Vip3Aa and Vip3Ae protoxins were effectively processed by midgut content extracts from the two Spodoptera species and the proteolytic activation did not produce a peptidase resistant core under these in vitro conditions. Digestion experiments performed with S. frugiperda chromatography-purified digestive serine peptidases showed that the degradation of the Vip3A toxins active core is mainly due to the action of cationic chymotrypsin-like peptidase. Although the digestion patterns of Vip3A proteins do not always correlate with toxicity, the peptidase stability of the 62 kDa core is in agreement with intraspecific differences of toxicity of the Vip3Aa protein.Journal of Insect Physiology 06/2014; 67. DOI:10.1016/j.jinsphys.2014.06.008 · 2.50 Impact Factor
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ABSTRACT: Each year billions of dollars are spent worldwide on insect control in agriculture . Despite this expenditure, up to 40% of a crop can be lost to insect damage, particularly in developing countries . Some of the most damaging insect species belong to the Lepidoptera, the second largest insect order comprised of moths and butterflies. The larval stage of moths cause major damage to an array of economically valuable crops including cotton, tobacco, tomato, corn, sorghum, lucerne, sunflower, pulses, and wheat . Until recently, broad spectrum chemical insecticides have been the primary control agent for agricultural pests, with about 40% targeted to the control of lepidopteran insects . Over the years the widespread use of pesticides has led to pesticide resistant insects, a reduction in beneficial insect populations and harmful effects to humans and the environment [5-8]. These problems have led researchers to develop different insect control strategies using both synthetic and natural molecules that are more environmentally friendly.
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ABSTRACT: The corn pith weevil Geraeus penicillus (Herbst) is occasionally found boring in corn stalks throughout the eastern and Midwestern United States. Injury caused by G. penicillus is not typically economical, but may be confused with that of the European corn borer Ostrinia nubilalis (Hubner), an important economic pest throughout the United States. During efforts to assess European corn borer infestations, we discovered G. penicillus in field corn in south‐eastern Pennsylvania, including hybrids genetically modified (i.e. Bt hybrids) to control European corn borer among other herbivore species. Our analysis across sites indicated that tunnels of G. penicillus were significantly more abundant in transgenic Bt hybrids than non‐Bt hybrids, but comparisons of Bt hybrids and their near isolines revealed mostly similar numbers of G. penicillus tunnels, suggesting other hybrid features might be affecting the distribution of G. penicillus. Tunnels of G. penicillus were equally distributed among the three transgenic trait packages represented in our study. In plants where we found G. penicillus, tunnels were more abundant in stalks free of European corn borer damage. Our report appears to be the first to note G. penicillus feeding in Bt corn hybrids. These findings are notable because they document insect damage in Bt hybrids that may be mistaken for European corn borer damage and may provide evidence of an insect herbivore proliferating following a mild winter or possibly even moving into competitor‐free space.Journal of Applied Entomology 03/2013; DOI:10.1111/jen.12051 · 1.70 Impact Factor