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| Cloning, Expression and Insecticidal Activity Analysis of Crystal2Ab32 (Cry2Ab32) Gene from Bacillus thuringiensis Strain |
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Abstract Aiming at enriching novel lepidoptera insecticidal genes, Bacillus thuringiensis strains BN23-5 was analyzed and identified by the PCR-restricted fragment length polymorphisms (PCR-RFLP) method, which indicated that crystal(cry) gene of cry2Ab-type gene was contained. According to the NCBI open cry2Ab-type gene encoding area, a pair of degenerate primer was designed. A full-length of 1 902 bp cry2Ab-type gene fragment, which was amplified by PCR with the degenerate primers using strain BN23-5 genome plasmid DNA as template, was inserted into the Escherichia coli expression vector pET-28a to obtain the recombinant plasmid pET-2Ab, and then transformed into E. coli BL21(DE3) cell. The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) verified that the target gene could express as 65 kD protein in E. coli BL21(DE3) strain by isopropyl-β-d-thiogalactoside (IPTG) induced at low temperature. What's more, expressed product of cry2Ab32 existed in bacteria precipitation after ultrasonic broken but not in the supernatant expressed product. The encoded protein composed 634 amino acid residues and shared a 96.5% sequence homology with Cry2Ab1 with 21 amino acids difference, which was designated as cry2Ab32 (GenBank No. KJ710647) by International Nomenclature Commitee of B. thuringiensis endotoxin. Bioactivity of cry2Ab32 gene expressed product was tested against Plutella xylostella, Ostrinia fumacalis, Laphygma exigua and Helicoverpa armigera by feeding method. The bioassay results indicated that expressed product of cry2Ab32 had a high insecticidal activity against P. xylostella, O. fumacalis and H. armigera, with median lethal concentration (LC50) of 8.99 μg/mL (95% confidence: 5.82~14.91 μg/mL), 19.34 μg/mL (95% confidence: 9.64~28.31 μg/mL) and 21.24 μg/mL (95% confidence: 13.35~30.32 μg/mL), respectively. However, Cry2Ab32 protein had no insecticidal activity against L. exigua, but could obviously inhibit the growth of L. exigua. In view of few previous reports about the high insecticidal activity of cry2Ab-type gene against O. fumacalis pest, the result was a major breakthrough for its prevention and control. The discovery of new gene cry2Ab32 will provide more choices for important agricultural pests prevention, and supply materials for further study of Cry2Ab-type protein structure and insecticidal mechanism.
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Received: 03 December 2014
Published: 13 April 2015
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