An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification ofcry1-Type Genes
ABSTRACTThecry1-type genes ofBacillus thuringiensisrepresent the largestcrygene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identifycry1-type genes using current methods because of the increasing number ofcry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes ofcry1-type genes was developed. This improved method was used to identifycry1-type genes in 20B. thuringiensisstrains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clusteredcry1-type genes and can be used to evaluatecry1-type genes in novel strain collections ofB. thuringiensis. Among the detectedcry1-type genes, we identified four novel genes,cry1Ai,cry1Bb,cry1Ja, andcry1La. The bioassay results from the expressed products of the four novelcrygenes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic againstPlutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity againstOstrinia furnacalis,Hyphantria cunea,Chilo suppressalis, andBombyx morilarvae and considerable weight loss activity againstHelicoverpa armigera.