Isolation, characterization and expression of a novel vegetative insecticidal protein gene ofBacillus 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.

Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria

“…Contrary to the above-described results, Gayen et al (60) found that a deletion of the first 200 N-terminal amino acids enhanced the insecticidal potency of the core active toxin ϳ2 to 3-fold against H. armigera, A. ipsilon, Spodoptera littoralis (Lepidoptera: Noctuidae), and Scirpophaga incertulas (Lepidoptera: Pyralidae). Similarly, in another study (49), a deletion of 33 amino acids from the Vip3Aa N terminus caused no loss of toxicity against S. litura, Plutella xylostella (Lepidoptera: Plutellidae), and Earias vitella (Lepidoptera: Noctuidae).…”

“…vip3 genes are ϳ2.4 kb in length, and they are normally carried on large plasmids (43,45), although in some cases, they have been proposed to be located in the bacterial chromosome (46). Many strategies for screening of B. thuringiensis isolates have been performed with the aim of isolating new vip3 genes (19,20,25,42,(46)(47)(48)(49)(50)(51)(52). At the time of writing of this review, there have been 54 vip3Aa, 2 vip3Ab, 1 vip3Ac, 4 vip3Ad, 1 vip3Ae, 3 vip3Af, 15 vip3Ag, 1 vip3Ah, 1 vip3Ai, 2 vip3Ba, 3 vip3Bb, and 4 vip3Ca genes reported (9).…”

“…Usually, the ef- (49,56,(58)(59)(60). There is generally agreement in that the last amino acids of the C terminus are critical for the activity and stability of Vip3 proteins, since their deletion, their replacement by nonconservative residues, or the addition of amino acids to the end of the protein completely abolishes protein activity (59,60) and increases susceptibility to proteases (57,58).…”

Mortality of Oryzophagus oryzae (Costa Lima, 1936) (Coleoptera: Curculionidae) and Spodoptera frugiperda (J E Smith, 1797) (Lepidoptera: Noctuidae) Larvae Exposed to Bacillus thuringiensis and Extracts of Melia azedarach

“…Thus, mortality due to the bacteria could be higher than that due to Cry proteins alone. The criteria to determine the insect specificity in the case of VIPs are not completely understood (Bhalla et al 2005). The entomopathogen includes other virulent factors, such as beta-exotoxins, alfa-exotoxins, hemolysins, enterotoxins, chitinases, and phospholipases (Höfte and Whiteley 1989;De Maagd et al 2003).…”

Biotechnological Approaches for the Control of Insect Pests in Crop Plants

“…This in turn has led to a search for new insecticidal proteins and their encoding genes that have commercial potential for plant protection [8,24]. They include -amylase inhibitors [25,26], vegetative insecticidal protein [27,28], chitinases [29] and protease inhibitors [30,31], as well as several other proteins directed to targets in the insect gut (Table 1).…”