The purified protein was stored at −20 °C in buffer 3. The activity of the Cry30Fa1 protein, obtained from the recombinant E. coli strain, was tested against P. xylostella (Lepidoptera), Helicoverpa armigera (Lepidoptera), and A. aegypti (Diptera). The larvae used in this study were reared in our laboratory. The bioactivity assays against P. xylostella and H.
armigera was performed as described by Song et al. (2003). The insecticidal activity of B. thuringiensis strains was assayed on the larvae of mosquitoes as described by Ibarra et al. (2003). Finally, the larvae were used for a treatment. Each Raf inhibitor treatment was replicated three times and its mortality was recorded after 72 h. The result of PCR amplification
showed that one special band, about 1.4 kb, was obtained using the primers S5un30/S3un30 (Fig. 1a). The amplification products were digested with the enzyme DraI and MspI, respectively. As shown in Fig. 1, the RFLP pattern, digested by the DraI contained three main bands (about 145, 450, and 800 bp, respectively), sizes which were similar to that of cry30Aa (Table 1). However, the RFLP pattern digested by the MspI revealed three main bands (about 250, 400, and 750 bp, respectively) that did not Maraviroc purchase coincide with the reported cry30Aa genes. Furthermore, this PCR product was cloned and sequenced and had the highest identity (84.8%) to cry30Aa1 when compared Calpain with the known cry30 genes. These results indicated that the strain BtMC28 contained a novel cry30-type gene. In order to obtain the full length of the novel cry gene, the Son-PCR upstream and
downstream strategies were performed using four nested specific primers. As Fig. 1b shows, the amplification products showed two to three bands in the first Son-PCR. After the second nested PCR, clear amplified bands could be seen at 800- and 1000-bp sites. These two bands were cloned and sequenced. The sequencing results indicated that the 5′ and 3′ ends of the cry30Fa1 gene were 829 and 947 bp, respectively. By assembling the known partial sequence of the cry30Fa1 gene with the 5′ and 3′ ends, the full-length sequence was obtained; it had about 3017 bp, which contains the ORF of 2064 nucleotides encoding a polypeptide of 687 amino acid residues with a predicted molecular mass of 77.1 kDa and an isoelectric point of 7.61. Sequence alignment analysis revealed that it corresponds to a putative Cry protein and was at maximum 74% homologous to that of Cry30Aa1 (Fig. 2). This novel cry gene was designated as cry30Fa1 by the B. thuringiensis Pesticide Crystal Protein Nomenclature Committee.
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