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Cloning and Detection of Antagonism-related Genes of Bacillus Cereus B-02

Author: DongZuo
Tutor: MaHuiQuan
School: Shandong University of Technology
Course: Biochemistry and Molecular Biology
Keywords: Bacillus cereus Transposition mutagenesis Tn917 southern hybridization TAIL-PCR
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 84
Quote: 0
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Abstract


Bacillus cereus (Bacillus cereus) B-02 separation greenhouse vegetable soil infected with Botrytis cinerea self, Zibo City, is an activity has a strong resistance to Botrytis cinerea (Botrytis cinerea) antagonistic bacteria. Explore strains the on Bo.cinerea mechanism of disease prevention, the use of the transposon tagging method to build the B-02 mutants antibacterial activity disappeared by TAIL-PCR method of insertion sites of transposable elements and their flanking genes aimed new antibacterial functionally related genes found in the clone, and analyze the sites where the gene or its flanking regulatory mechanism of the gene on the phenotype. Extraction of Bacillus subtilis (from Bacillus subtilis) PY143 carrying transposon Tn917 non shuttle resistance temperature-sensitive plasmid of pTV1, electric shock transformed pTV1 plasmid to wild Ba.cereus B-02 strain, in the voltage 9.0-12.5kv/cm resistor 200Ω capacitance of 25μF electroporation conditions with chloramphenicol resistance and can be stable, continuous replication of 17 transformants, and plasmid electrophoresis and PCR analysis confirmed. The transformants in the 44.5 ℃ continuous high temperature mutagenesis, pTV1 plasmid eliminate, Tn917 inserted into the Ba.cereus B-02 genome was screened with erythromycin and lincomycin resistance, but the loss of chloramphenicol resistance the transposition mutants 1670, in this experiment, the average transposition efficiency of 4.29 × 10 -4 , subculture prove mutant strain has genetic stability, indicating that Tn917 inserted fragment in most mutant strains can be stably inherited prove pTV1 Tn917 fragment inserted into Ba.cereus B-02 genome by PCR. Further determination of these mutants on inhibition of Bo.cinerea, the final screening has been the loss of immunity to the pathogen mutants, named B-02-T. Randomly selected 6 mutant strains containing portion Tn917 fragments DIG-labeled probe and EcoR Ⅰ digesting its genome southern hybridization, the results confirmed that the genome of the mutant strains and only a single Tn917 insert, and can the random mutagenesis Ba.cereus wild strains to produce a mutant strain in a different point mutation. TAIL-PCR method Tn917 insertion site flanking sequences were obtained Tn917 insertion site on the downstream of the two gene fragments cloned from the mutant strain B-02-T, by the removal of contamination of the carrier, obtained after a period of Preparation gene splicing 2705bp gene sequence, after the blast than found Bacillus plasmid sequence homology, the homologous sequences expression of protein hypothetical protein, suggesting possible new genes associated with antibacterial activity.

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