Cloning And Functional Study Of Insecticidal Crystal Protein Gene From Bacillus Thuringiensis New Strains

The pest resistance has received much attention with the long-term use of Bacillus thuringiensis insecticides.In a variety of resistance management strategy,screening new Bt strains and new insecticidal toxins is an effective method for delaying pest resistance.In this study,we collected 118 soil samples from different parts of our country and isolated five Bt strains by temperature screening method.It showed typical diamond crystal under microscopic observation.PCR analysis showed that the strains contained cry1 type gene.The strain named as Bt 6618 was singled out and characterized.Its parasporal crystal was observed through microscopic morphology and 16S rRNA gene identification.SDS-PAGE and LC-MS/MS assay indicated the strain expressed 13 0-kDa protein which constituted Cryl Ae and Cryl Ac toxin.Sequence analysis showed that the crylAc gene of the strains was identical to crylAcl gene published sequences.The coding sequence of Cry1Ae toxin remained to be obtained.The spore-crystal mixture of the strain had high toxicity towards Helicoverpa armigera larvae with an LC50 value of 55?g/mL.In addition,we have studied insecticidal genes of the known Bt subsp.kyushuensis strain 4U1.We observed crystal morphology of this strain by scanning electron microscope.It produced oval crystals and expressed 13 0-kDa toxin protein by SDS-PAGE analysis.PCR analysis showed that the strains contained cry7 type gene and cry8 type gene.Mass spectrometry analysis showed the toxin component of this strain contained Cry7 and Cry8 proteins.It showed that the cry8 gene of this strain was identified to the CDS sequence of ARP110 strain by cloning and analyzing the gene.This gene was the first found in the strain.Toxicity bioassay showed that spore-crystal mixture of the strains have low virulence towards H.armigera larvae.The toxicity to other insects remains to be analyzed in future.The isolated Bt strain with high toxicity in this study will not only enrich reserves,but also provide resources for the development of new efficient biological pesticide.