Characterization of insecticidal crystal protein cry gene of Bacillus huringiensis from soil of Sichuan Basin, China and cloning of novel lotypes cry gene

Abstract

Sichuan Basin situated in the west of China, the fourth-largest basin of China, is a special area with complicated geomorphology (mountain, pasture, gorge, virgin forest, highland, hurst, glacier, and plain), and contains a rich and unique biodiversity. In order to describe a systematic study of cry gene resources from Bacillus thuringiensis (Bt) strains of different ecological regions in Sichuan Basin, a total of 791 Bt strains have been screened from 2650 soil samples. The analysis of the cry genes was based on the method of PCR-restriction fragment length polymorphism (PCR-RFLP). cry1, cry2, cry3, cry4/10, cry9, cry30, and cry40-type genes were found in this basin. Strains containing cry1 genes were the most abundant in our collection (66%), and twenty-one different cry1-type gene combinations were found. Bt strains harboring cry2 genes were the second most abundant (39.5%), and the strains containing cry3, cry9, cry4/10, cry30, and cry40 genes were found in 2.5, 3.5, 4.2, 4.2, and < 1%, respectively. Furthermore, several novel haplotypes cry genes were found, and the full-length sequences of three novel cry genes were obtained, which were designated as cry52Ba1, cry54Aa1, cry30Fa1 by the B. thuringiensis Pesticide Crystal Protein Nomenclature Committee, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) assay of 80 strains which did not produce any PCR products indicated that these strains may harbour potentially novel Cry proteins. All these researches mentioned above revealed that the diversity and particularity of cry gene resources from B. thuringiensis strains in Sichuan Basin.