Screening Of Bacillus Thuringiensis And Cloning And Sequence Analysis Of Gene Coding Parasporal Crystal

Bacillus thuringiensis (Bt), one of the microorganisms widely found in nature, canproduct parasporal crystal with insecticidal activity against to over200insects, such asLepidoptera, Diptera, Coleoptera, Orthoptera, Nematodes etc. which is the main componentof the bio-pesticides now used in the world. have an effect on The parasporal crystals fromdifferent strains used in the world have the different insecticidal characteristic, and the crygenotype coding the parasporal crystal have a total of70.This paper focused on the researche of the screening and identification of B.thuringiensis strains,, cloning and sequence analysis of the gene coding parasporal crystal.With strains of Bacillus thuringiensis subsp. kurstaki CGMCC1.1752(genotype ascry1Ac) and subsp israelensis CGMCC1.1754(genotype as cry4A, cry4B) as control, coupledobservation and partial gene cloning of parasporal crystal, the method of strains screeningwere developed. Strain TS3was screened from a total of12soil samples collected fromMedicinal Botanical Garden and Tsinghua University using the aboved method. The isolatedstrain TS3was a member of B. thuringiensis based on the phylogenetic analysis of16S rRNAgene and chemotaxonomic characterization performed with API20E, API50CH, andAPIZYM.The cloning and sequence analysis of partial cry gene from strain TS3was belong tocry8genotype. According the cry8gene sequences from NCBI(http://www.ncbi.nlm.nih.gov/), the primers were designed, and the full-length sequence(3642-bp) of cry gene was PCR amplified from TS3. The cry gene belong to cry8I encodes a1213amino acids with a calculated molecular weighe of about137kDa. The second structureof Cry8I protein from TS3was calculated with ProtScale and HNN in swiss-model program.With B. thuringiensis subsp. kurstaki CGMCC1.1752as indicator, the fermentationcondition including carbon and nitrogen sources for production of parasporal crystal wasperformed.