Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (11): 2232-2239.doi: 10.3864/j.issn.0578-1752.2013.11.006

• PLANT PROTECTION • Previous Articles     Next Articles

Construction of Bacillus subtilis Bs916 Mutant Libraries by Transposon Tagging and Cloning the Genes to the Organism’s Anti-Bacterial Activities

 ZHOU  Hua-Fei, LUO  Chu-Ping, WANG  Xiao-Yu, ZHANG  Rong-Sheng, CHEN  Zhi-Yi   

  1. 1.College of Plant Protection, Nanjing Agricultural University, Nanjing 210095
    2.Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2013-01-08 Online:2013-06-01 Published:2013-03-07

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Abstract: 【Objective】The objective of this study is to construct Bacillus subtilis Bs916 mutant library by introducing transposon into Bs916 genomic DNA, screen mutants which to appear enhanced or decreased apparently in the inhibition zone to Xanthomonas oryzae pv. oryzicola (Xooc), and to obtain the genes which may be related to anti-bacterial activities changed by cloning the DNA sequence around the transposon inserted sites. 【Method】The pMarA shuttle vector which contained transposon TnYLB-1 was introduced into B. subtilis Bs916 through chemical transformation method and build the random inserted mutant library. The enhanced or decreased anti-bacterial activity mutants screened from the library by in vitro assay against Xooc and the mutants were verified by the PCR and Southern Blot. The genes around the insertion sites were amplified by the Inverse PCR and sequence analyzed by bioinformatics.【Result】The random inserted mutant library of B.subtilis 916 was constructed successfully by using TnYLB-1 transposon. The PCR and Southern Blot results showed that the transposon was inserted into Bs916 genomic DNA successfully, about 85% mutants were inserted by a single copy. More than 30 mutants whose antibacterial activities changed against Xooc strongly enhanced or sharply decreased were screened. Twenty-one genes around the insertion site were cloned from the mutants above and sequenced. The bioinformatic analysis results showed that these genes were related to competence development, bio-film formation and secondary metabolites synthesis.【Conclusion】A random transposon inserted mutant library of B. subtilis Bs916 was constructed successfully. These cloned genes in mutation points from the library to Xooc with inhibitory effect significantly changed. It was speculated that these genes may be related to synthesis and regulation of the anti-bacterial compound in B. subtilis Bs916.

Key words: Bacillus subtilis , mutant , Xanthomonas oryzae pv. oryzicola , antibacterial gene , clone

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