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Annals of Microbiology
2015

Diversity of insecticidal crystal protein genes of Bacillus thuringiensis isolated from soil and cloning of novel haplotypes of cry genes

Zonglan Yu, Li Gong, Qiao Li, Ganghui Huang, Liye He, Ping Li, Aiping Zheng

Rice Research Institute, Sichuan Agricultural University, Wenjiang, Sichuan, 611130, China.

Abstract

Bacillus thuringiensis (Bt) strains used in this study were isolated from forestland soil in Chengdu, China. Sodium acetate-antibiotic treatment was used to isolate the strains, and scanning electron microscopy was used to identify crystal morphology. Diversity was observed in the protein and cry gene profiles, and most of the isolated Bt strains produced 130, 90, 80, 70, 66, and 45 kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Polymerase chain reaction (PCR) analysis of these isolates identified 14 cry genotypes and 30 different genotype combinations. Several novel cry fragments were discovered using PCR restriction fragment length polymorphism (RFLP) analysis, and five novel full-length cry genes—cry1Hc1, cry1Ie5, cry30Ea4, cry54Ba2, and cry56Aa4—were cloned. The analysis of insecticidal activity indicated that the majority of the isolates had high activity against Ostrinia furnacalis larvae; the cloned novel genes cry1Hc1 and cry1Ie5 conferred high activity against Ostrinia furnacalis larvae. Forty isolates did not yield PCR products, and eight of these had activity against Ostrinia furnacalis larvae. The results suggest that the genetic resources are rich in these isolates, which have the potential for future use as insecticides.

Keywords: Bacillus thuringiensis, PCR-RFLP, SDS-PAGE, cry gene, Novel gene haplotypes.

 
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