Novel Bacillus thuringiensis ?-endotoxin active against Locusta migratoria manilensis.

ABSTRACT: A novel ?-endotoxin gene was cloned from a Bacillus thuringiensis strain with activity against Locusta migratoria manilensis by PCR-based genome walking. The sequence of the cry gene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The ?-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no. EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 ?-helices (domain I); 213 residues forming three antiparallel ?-sheets (domain II); and 134 residues forming a ?-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of the cry gene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed in Escherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC??s) were 8.98 ?g/ml for the expressed Cry7Ca1, 0.87 ?g/ml for the activated toxin 1, and 4.43 ?g/ml for the activated toxin 2. The ?-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.

SUBMITTER: Wu Y 

PROVIDER: S-EPMC3126473 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature