Sceening Of New Bacilluds Thuringiensis Strains With High Insecticidal Activity And Gene-types Identification

Bacillus thuringiensis (Bt) is a cporc-forming gram-positive bacterium. During sporulation, the intracellular insecticidal crystal proteins (Cry proteins) are produced as phase-bright inclusions. These proteins are toxic to insect larvae in the orders Lepidoptera, Diptera, and Coleoptera. The Cry protein from Bt has been developed as one of the most successful biological agents in industry to control insect pests. To obtain novel Bt strains for production of Cry proteins, the isolation of fourty seven Bt isolates was carried out from soil samples and diseased larvae, respectively in Hebei Province. Insecticidal activity from 11 isolates of them against neonats of spodoptera exigua, Mythimna separata, Helicoverpa armigera, Ostrinia furnacalis and Plutells xyiostella was measured by bioassay in incorporating a suspension containing tenfold serial dilution of purified inclusion into the wheat germ diet. The results showed that two of the 11 isolates, T4-3 and CYZ-13, appeared high toxicity with the LC50 value of 1.5 X 106 and 2.10 X 106 cell/ml, respectively.Four pairs of universal primers (K5un2/K3un2, K5un3/K3un3, S5un2/S3un2, and S5uni/S3uni) were designed. The products of PCR amplification with the four pairs of primers above, 1.6kb, 1.4kb, 1.2kb, and 1.6kb fragments, were digested by using enzymes of Pst1/Xbal, EcoR1/ Pstl, Msp1/Mval, and Banll91/Banl, respectively. Based on their RFLP patterns and specific fragments, the results of PCR-RFLP analysis showed that strain T4-3 was identified containing cry1Aa, cry 1 Ac, crylAg, cry2Ac, and cry1Ia1 genes and strain CYZ-13 contains crylAc, crylBc, and cry2Ab genes. Based on biochemical analysis showed that T4-3 strain was considered as B. thuringiensis subsp. Subtoxicus and CYZ-13 strain belongs to B. thuringiensis subsp. kurstaki.