Abstract: 【Objective】The objective of this study is to identify the functional genes, with their expressed proteins, and insecticidal spectrum of Bacillus thuringiensis strain CPB012. This will be important for effective application of CPB012 in pest control in the future.【Method】CPB012 was cultured at 30℃ on LB plates for two to three days and the bacterial cells were stained with phenol magenta for microscopy observation of the protein crystals. cry1-cry10 were identified with the polymerase chain reaction-restriction frame length polymorphism (PCR-RFLP) technique, namely, a universal primer pair for a group of genes was designed and used to amplify the genes with PCR. The amplified genes were then catalyzed with restriction enzymes and thus the polymorphism of the insecticidal genes were decided. On the other hand, specific primers were designed for identifying sub-group or genes (such as cry1A, cry2Aa. etc.) in groups cry1-cry10 via PCR amplification and then these genes were sequenced and compared. cry1-cry40 and vip groups were identified with PCR through the design of primers. The crystal proteins were extracted, respectively, from the bacterial cells cultured on LB plates for 24, 47, 72 and 96 hours and SDS-PAGE chromatographic fingerprint analysis was applied to demonstrate different insecticidal proteins. The lethality of Bombyx mori (Lepidoptera), Leptinotarsa decemlineata (Cleoptera) and Bactrocera dorsalis (Diptera) by CPB012 was measured in the laboratory, the cooperative effects of CPB012 and MJ07 strains of Beauveria bassiana were tested under both laboratory and field conditions. 【Result】Continuous microscopic observations found that CPB012 produced spherical and cubic protein crystals in blastosporic cells. Gene type analysis showed that B. thuringiensis CPB012 contained cry1Aa, cry1Ia, cry2Aa, cry2Ab and vip3A genes. These genes were shown via SDS-PAGE chromatographic fingerprint to code crystal proteins of about 135, 80, 70, and 85-90 kD, respectively. Also, the bacterium was demonstrated to contain two new crystal proteins which were 30 and 50 kD. Laboratory tests showed that CPB012 infected B. mori, B. dorsalis and L. decemlineata caused more than 76% deaths of each pest. After a mixture of B. thuringiensis CPB012 and B. bassiana MJ07 (mixed at 0.5: 0.5) was applied in a laboratory test, the cumulative mortality rate of 2nd-instar L. decemlineata larvae was 96.5%, which was significantly higher than that of individual CPB012 (83.3%) and that of MJ07 (90.1%). The LC₅₀ calculated from the multiple regression equation was 1.10×10⁷ cfu/mL and the CTC was 254, which indicated the combined use of the two biocontrol agents increased pest control efficacy markedly. Under the local field condition, the mixture of CPB012+MJ07 treatment also improved the pest control effect markedly; the cumulative mortality of the 1st- and 2nd-instar larvae were 47.5% and 43.8%, respectively, which were significantly higher than those of separate CPB012, MJ07 and commercial Bt preparation.【Conclusion】The insecticidal function of B. thuringiensis CPB012 was decided by cry1Aa, cry1Ia, cry2Aa, cry2Ab and vip3A and these genes encoded insecticidal proteins of 135, 80, 70, and 85-90 kD. This bacterium had wide spectrum of insecticidal activity which was shown by high death rates of B. mori, B. dorsalis and L. decemlineata induced by CPB012 treatment. The insecticidal efficacy was significantly improved by combined application of CPB012 of B. thuringiensis and MJ07 of B. bassiana. Therefore, CPB012 is thought of as a wide-spectrum biocontrol agent and will be of potential significance and great value in developing effective bio-insecticide.

Key words: Bacillus thuringiensis CPB012, functional genes, insecticidal crystal proteins, Leptinotarsa decemlineata, Beauveria bassiana MJ07, biological control