Identification and characterizarion of novel vip3-like genes inBacillus thuringiensis strains from a Spanish collection


Abstract: A total of 400 strains of Bacillus thuringiensis (Bt) have been screened to identify thepresence of vip3-like genes using a PCR-restriction fragment length polymorphism (PCR-RFLP)strategy to detect novel vip3 genes. This allowed identification of vip3A known genes in ~80% ofthe strains, whereas three new RFLP patterns revealed the presence of three novel vip3-like genesin three different Bt strains (B5.3, B8.1, B8.3). PCR amplicons from B8.1 and B8.3 strainsproduced fragments of 1646 and 1642 bp, respectively that were cloned and sequenced. Thesetwo fragments shared homologies of ~77% with known vip3 genes suggesting they may be novelvip3 genes. The strain B5.3 amplicon was 1620 bp and showed 91% homology with vip3Aa1gene. The full-length sequence of this vip3A-like gene was obtained and found to be a newvip3Ag variant and was designated vip3Ag4 by the Bt Toxin Nomenclature Committee. This geneis 2364 bp in size, encodes a 788 amino acid (88 kDa) protein and shares between 81 and 91%homology with known vip3A genes. Furthermore, vip3Ag4 was successfully cloned in pET-28b(+) and expressed in Escherichia coli BL21(DE3). The protein obtained was toxic againstChrysodeixis chalcites, Helicoverpa armigera, Spodoptera exigua, S. frugiperda and S. littoralis.

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