title: Novel Bacillus thuringiensis δ-endotoxin active against Locusta migratoria manilensis.
abstract: A novel δ-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 δ-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 α-helices (domain I); 213 residues forming three antiparallel β-sheets (domain II); and 134 residues forming a β-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 (LC₅₀s) were 8.98 μg/ml for the expressed Cry7Ca1, 0.87 μg/ml for the activated toxin 1, and 4.43 μg/ml for the activated toxin 2. The δ-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.
authors: Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China.
authors: Yan
authors: Y
authors: Wu
authors: Cheng-Feng
authors: CF
authors: Lei
authors: Dan
authors: D
authors: Yi
authors: Peng-Ming
authors: PM
authors: Liu
authors: Mei-Ying
authors: MY
authors: Gao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000472:pathogenicity*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D015153:Blotting, Western; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D004926:Escherichia coli / Q000235:genetics; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D006651:Histocytochemistry; D048188:Locusta migratoria / Q000187:drug effects*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016019:Survival Analysis
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis