[Characterization of insecticidal crystal protein cry gene of Bacillus thuringiensis from soil of Sichuan Basin and cloning of novel holotype cry gene].

OBJECTIVE

In order to systematically investigate the cry gene resources from Bacillus thuringiensis (Bt) in different ecological regions in Sichuan Basin and further clone novel cry genes.

METHODS

We used the methods of microscopic and scanning electron microscopic observation, PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and test of insecticidal activities to research Bt strains collected in this basin.

RESULTS

We screened 791 Bt isolates from 2650 soil samples, and found cryl, cry2, cry3, cry4/10, cry9, cry30, and cry40-type genes in this basin. Strains containing cryl genes were the most abundant in our collection (66%), and 21 different cry1-type gene combinations were found. Furthermore, several novel holotype cry genes were found and the full-length sequences of 3 novel cry genes were designated as cry54Aa1, cry30Fa1, and cry30 Ga1 by B. thuringiensis Pesticidal Crystal Protein Nomenclature Committee. The results of insecticidal activities showed that Sichuan Basin harbored Bt isolates with insecticidal activities. SDS-PAGE assay of 80 strains without PCR products indicated that these strains may harbor potentially novel cry proteins gene.

CONCLUSION

The diversity and particularity of cry gene resources in Sichuan Basin have important meanings in theories and practices.