| Food and ecological environment is a necessity of survival for human beings. Due to the extensive use of chemical pesticides, strengthen pest resistance, serious problems of pesticide residue and environmental pollution, human life is seriously threatened. Bacillus thuringiensis is one of microbial pesticides of most studied and the most widely application prospects in abroad.It has the unparalleled advantage of chemical pesticides do not.This study collected2soil mix samples from Danzhou campus of Hainan University by using five-point sampling. Using sodium acetate-antibiotic isolated13parasporal crystals containing Bacillus thuringiensis from soil sample. Its form has diamond-shaped and round crystals. The total detection rate was9.26%. Which heat and add the highest detection rate of penicillin treatment was9.26%, followed by heating and adding sodium acetate, sodium acetate and heated and penicillin and heat treatment.Picked isolated single colonies of Bacillus thuringiensis13inoculated in LB liquid medium,30%by microscopic crystals fall off after fermentation broth dilution10-1,10-2,10’3three concentrations of2or3in star larvae of the cotton bollworm bioassay, after screening has been compared with five pairs of cotton bollworm high activity strains, respectively, A322, C1-3, C231, B413and D323, which the A322strain highest insecticidal activity of bollworm insecticide rate of40%after72h, respectively,36.7%,26.7%,26.7%and20%. High-speed centrifugation to collect crystals bollworm parasporal be rescreening,3d check this five strains of Helicoverpa armigera Hubner its insecticidal dilution rates were30,26.7,20,16.7and16.7. The results show:A322strains highest insecticidal activity, is to be screened strains in this study.Appraisal the morphology physiological and biochemical characteristics,16SrDNA and genotype cry gene amplification of strain A322.The results show that: the strain vegetative rod-shaped, rounded at both ends, or as a single chain. Both ends of the crystal, the crystal can produce diamond and round:cylindrical-type colony morphology, color white, matte and rough, jagged, to the surrounding scattering; physiological and biochemical results and characteristics of Bacillus thuringiensis consistent; The highest similarity of16SrDNA sequence of strain with Bacillus cereus ATCC14579.1is99%. Initially identified result of strain A322is Bacillus cereus subtilis;using one pair of universal primers (SH-1and SH-2) to amplify the gene sequencing results by comparison with Bacillus thuringiensis strain HTS-S-38Cryla pattern similarity several Bacillus sequences such thuringiensis gene KF980886.1reached99%, A322were identified as strains of Bacillus thuringiensis.According to the measured result of strains BtA322in liquid culture in LB growth curve shows:strain A3220-4h in the incubation period,4-24h in the logarithmic phase,24-36h entering the stable period, after36h entering the decline phase that parasporal crystals produced period. Basic medium is liquid LB medium, the results of Single factor and orthogonal experiment to optimize its culture medium and fermentation conditions:strain A322optimum medium was0.5%soybean powder,0.5%yeast extract,0.5%glucose,0.5%tryptone,0.2%calcium carbonate,0.1%potassium dihydrogen phosphate,0.1%magnesium sulfate. And to determine the optimum fermentation conditions; pH7.0, fermentation temperature is30℃, culture time was20h, inoculum size3%, aeration rate35ml/250ml triangle bottled liquid volume. Growth of bacteria after the optimization of OD600value was increased by21.9%than that before optimization OD600. |
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