An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1 -Type Genes

Abstract

ABSTRACT The cry1 -type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1 -type genes using current methods because of the increasing number of cry1 -type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1 -type genes was developed. This improved method was used to identify cry1 -type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1 -type genes and can be used to evaluate cry1 -type genes in novel strain collections of B. thuringiensis . Among the detected cry1 -type genes, we identified four novel genes, cry1Ai , cry1Bb , cry1Ja , and cry1La . The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella , whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis , Hyphantria cunea , Chilo suppressalis , and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera .