Isolation Of Bacillus Thuringiensis Strains From Coalfields And Identification Of Novel Insecticidal Genes

Bacillus thuringiensis(Bt)is an insect pathogenic bacterium that has been widely used in biological control of agricultural insect pests.Transgenic Bt producing insecticidal crystal proteins is the most important method to control the cotton pest Helicoverpa armigera.However,resistance to transgenic Bt appeared in the field and it is critical to find novel Cry toxins to avoid insect resistance.However,due to some factors,like man-made interruption and pesticide residue,it is difficult to isolate novel Bt strains from field sources.Therefore,it is important to isolate new Bt strains from special habitats.We have isolated two novel Bt strains from 315 samples of coalfields,named BRC-LBR1 and BRC-LBR2.We used physiological and biochemical tests to determine their biological characteristics,showing that the two isolates can ferment glucose and polychrome,hydrolyze starch,produce arginine decarboxylase,voges-proskauer test was negative for both strains,and urease test was positive only for BRC-LBR1.Insecticidal activity of BRC-LBR1,BRC-LBR2 as well as Bt subsp.kurstaki HD73 standard strain were carried against H.armigera.Both isolates showed insecticidal activity against H.armigera,and BRC-LBR1 showed higher toxicity than HD73 strain.Cry genotypes of BRC-LBR1 and BRC-LBR2 were identified by PCR,with 11 pairs of universal primers.Both isolates have the cry1 gene,which was cloned and sequenced.Homology analysis showed that cry1 gene of BRC-LBR1 was up to 99%similarity with the cry1Ac gene,by contrast,cry1 gene of BRC-LBR2 had lower similarity with cry1Ka gene,was up to 98%.Moreover,sequencing data showed that BRC-LBR2 encodes two insecticidal protein genes.The two genes were submitted to NCBI with the GenBank Accession No.:KU761577 and KU761578,and were named crylBe5 and vip3Ad6 by International Bt Naming and Classification Committee.LC-MS mass spectrometry from the parasporal crystal proteins of BRC-LBR2 showed that the crystal protein expressed by BRC-LBR2 was 139 kDa,and identified as Cry1Be,consistent with the sequencing results.Functional domains,secondary structure and three-dimensional structure of the two proteins were also analyzed.Generally,our results show that new Bt strains can be isolated from the coalfields and posses novel toxin genes against H.armigera,that can be used to develop effective biological control agents for crop pests.