Isolation And Identification Of Bacillus Thuringiensis And Cloning Of Insecticidal Genes

Bacillus thuringiensis(Bt) was a spore forming Gram-positive bacteria, it haddeveloped into the largest global production, the application area of Bt was the mostextensive microbial pesticides. Because of the long-time use of Bt formulation, a series ofproblems, such as resistance and narrow-spectrum of insecticide. It has important practicalsignificance that required us to discovery of new genes and explores a new activity.Seventy two Bt isolates were obtained from756soil samples which were collectedfrom the Wuyuezhai Mountains of Hebei province by using the temperature method.Several shapes of crystals protein were observed by optical microscope, such as spherical,diamond, ovoid, square, irregular shape, et al. All showed the great diversity of Bt strainsin Wuyuezhai Mountains.Seventy two Bt strains were identified by using of PCR-RFLP method, and PCRanalysis using novel general and specific primers for cry genes. The result showed thatthirteen kinds of cry gene, cry1, cry2, cry3, cry4, cry6, cry8, cry9, cry11, cry18, cry22,cry26, cry32and cry1I, were found in72Bt strains. Among them,38strains harbored cry8genes, The cry genes in other8Bt strains were not identified, possibly include theunknown genotypes, and need to draw supports from other methods to more research.The result of SDS-PAGE analysis showed that both of the strains expressed130kDa,90kDa and50kDa protein bands in the first. The strains which contain cry1genes andcry2genes mostly expressed130kDa and60kDa protein bands; strains contain cry1genesor cry2genes expressed130kDa protein band and formed diamondoid crystals mostly;strains contain cry8genes expressed130kDa bands mostly and formed spherical or ellipticcrystals; The strains which contain cry3and cry8genes mostly expressed130kDa proteinbands; Bt strains that contain cry22genes mostly expressed90kDa protein band; Strains ofwhich genetypes were not identified expressed various protein bands.The WYZ-54was highly toxic to P.aenescens larvae which the corrected mortalitywas80%, it harbored cry1,cry2and cry1I genes only, The fragment(1522bp) of cry genecloned from strain WYZ-54had99%sequence high-comparability with the Cry1Ia-likeprotein (cry1Ia) gene（FJ617448.1）. The strain FPT-25showed highly toxic to Anomala corpulenta larvae, The type ofinsecticidal genes in this Bt strains was unknown, SDS-PAGE analysis showed that thestrain expressed80kDa and50kDa protein bands. Three pairs of primers were used toamplificate its conservative sequences, and1900bp genetic fragments were gained andsequencing. The Blast analysis showed that it sequence had no high-similarity with keownBt genes in GenBank.All the isolates carried on bioassay to P.xylostella, P.aenescens, H.oblira, andA.corpulenta. The result showed that, eleven strains were highly toxic to P.xylostella, thecorrected mortality of nine of them were100%. thirteen strains were highly toxic toP.aenescens. four strains showed highly toxic to Holotrichia oblita larvae. the correctedmortality of them were60%、80%、60%、80%, the result of LC50showed the insectdisinfestation effect of WYZ-12and WYZ-30were higher than standard strain HBF-18,the other two were lower.