枯草芽胞杆菌Bs916突变体库的构建和抑制水稻 细菌性条斑病菌相关基因的克隆

doi:10.3864/j.issn.0578-1752.2013.11.006

摘要/Abstract

摘要： 【目的】采用转座子标签技术构建枯草芽胞杆菌Bs916突变体库，从突变体库中筛选对水稻细菌性条斑病菌抑菌效果发生显著变化的菌株并克隆插入位点的基因，获得生防菌Bs916中与抑制细菌活性可能相关的基因。【方法】携带转座子TnYLB-1的穿梭载体pMarA通过化学转化方法转化至枯草芽胞杆菌Bs916菌株，构建随机插入突变体库。通过平板抑菌试验从突变体库中筛选对水稻细菌性条斑病菌抑菌效果得到显著提高和下降的突变体；并采用PCR和Southern Blot验证转座子是否成功插入到Bs916染色体基因组上，利用反向PCR技术克隆转座子插入位点基因、测序并进行生物信息学分析。【结果】成功构建了枯草芽胞杆菌Bs916的转座子随机插入的突变体库；PCR和Southern Blot结果表明转座子成功的插入到染色体基因组上，约85%突变体以单拷贝形式插入；筛选出30株对水稻细菌性条斑病菌抑制效果发生显著变化的菌株，克隆到21个突变体插入位点的基因序列并测序。生物信息学分析结果表明这些基因与感受态形成、生物膜形成和次生产物代谢相关。【结论】成功构建了Bs916突变体库，克隆到该库中对水稻细菌性条斑病菌抑菌效果显著变化菌株的突变位点基因，这些基因推测可能与枯草芽胞杆菌Bs916中的抑制细菌化合物的合成代谢及调控相关。

关键词: 枯草芽胞杆菌 , 突变株 , 水稻细菌性条斑病菌 , 抑菌基因 , 克隆

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

周华飞, 罗楚平, 王晓宇, 张荣胜, 陈志谊. 枯草芽胞杆菌Bs916突变体库的构建和抑制水稻细菌性条斑病菌相关基因的克隆[J]. 中国农业科学, 2013, 46(11): 2232-2239.

ZHOU Hua-Fei, LUO Chu-Ping, WANG Xiao-Yu, ZHANG Rong-Sheng, CHEN Zhi-Yi. Construction of Bacillus subtilis Bs916 Mutant Libraries by Transposon Tagging and Cloning the Genes to the Organism’s Anti-Bacterial Activities[J]. Scientia Agricultura Sinica, 2013, 46(11): 2232-2239.

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