Isolation And Identification Of Bacillus Thuringiensis And Cloning Of Insecticidal Genes From Damaoshan Mountains

Bacillus thuringiensis (Bt) has been widely used in the world as a kind of bio-control microbial. For harmless to persons, animals and the non-target insects, Bt possess huge economic value and ecological value. In order to resolve a series of problems just like pest resistances occurrence or narrow-spectrum of insecticide, screening of new Bt strains with high insecticidal activity become very important.In this research, 76 Bt stains were isolated from total 784 soil samples which were collected from the Damaoshan Mountains of Hebei province by temperature- antibiotics screening method. Microscopic observation results showed that the spore crystal include diamond, spherical, square, ovoid, irregular shape, et al. The great diversity and richness of Bt strains had been proved in Damaoshan Mountains.In this research, 76 Bt stains were identified by using of PCR-RFLP. The results showed that all the stains contained 8 kinds of genotypes: cry1, cry2, cry3, cry8, cry9, cry26, cry1I and vip, and the cry8 genes were found in 34 strains accounted for the biggest proportion, the cry1 gene were found in 12 strains, the cry2 gene were found in 5 strains, the cry3 gene were found in 2 strains, the cry9 gene were found in 7 strains, the cry26 gene were found in 6 strains, the cry1I gene were found in 11 strains and the vip gene were found in 3 strains. In the gene combination, 7 strains owned both cry8 and cry9, 5 strains owned both cry1 and cry2, and 6 strains owned cry8 and cry26. Besides, 134 stains which were not identified genetypes, possibly include the unknown genotypes, and need to draw supports from other methods to more research.SDS-PAGE protein crystals analysis results showed that the strains which contained cry1 genes mostly expressed 130kDa protein; the strains which contain cry1 genes and cry2 genes together expressed 130kDa and 60kDa protein; the strains which contain cry8 genes expressed 130kDa protein; the strains which contain cry8 genes and cry9 genes mostly expressed 130kDa and 80kDa protein; the strains which contain cry3 genes and cry8 genes mostly expressed 130kDa protein. The size of protein which were expressed in the strains had not identified genotype were diverse.Bioassay of 76 Bt strains against larva of Holotrichia oblira, A.corpulenta, H.parallela, Plutella xylostella and Heliothis armigera was conducted. Results showed that 10 strains had insecticidal activity to lepidoptera insect with the mortality rate which was 100%, 4 strains were toxic to Holotrichia oblira insect with the mortality rate which was past 70%. The strain TDM-19 had high activity against A.corpulenta, and the 100% mortality was gained after 14 days. The LC50 was 2.4943×108 spores/ml to A.corpulenta, and the insect disinfestation effect is higher than standard strain HBF-1. The strain expressed 97kDa, 66kDa, 45kDa protein, but not the 130kDa protein which had high activity against A.corpulenta. The expectations fragments had not been gained by using the general primer to identificate its genotype. The degenerate primers OL1/OL5 were used to amplificate its conservative sequences, and 900 bp genetic fragments were gained. The fragment of 928bp cloned from the strain TDM-19 had no high-comparability with Bt genes known in GenBank.Full-length of cry8Ca3 gene were cloned from the strain FB-32. The genetic encoding area was 3483 bp, including 1160 amino acids, and the molecular weight was 130.32 kDa. The isoelectric point was 4.74 for weak acidity amino acids. The gene had been nominated as cry8Ca3 by International Nomenclature Committee of Bt as GenBank accession numbers EU625349.