绿针假单胞菌YL-1抗细菌活性相关基因的克隆和分析

绿针假单胞菌YL-1对植物多种重要病原细菌和病原真菌有较强的抑制作用。采用转座子插入突变技术电转化绿针假单胞菌YL-1,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3种病原细菌荚壳伯克霍尔德菌Burkholderia glumae、解淀粉欧文氏菌Erwinia amylovora和胡萝卜果胶杆菌Pectobacterium carotovora抑菌效果显著下降的突变体,并采用PCR和Southern Blot验证转座子是否成功插入到YL-1染色体基因组上。利用质粒拯救技术克隆转座子插入位点基因、测序和生物信息学分析。结果表明:本研究成功构建了绿针假单胞菌YL-1随机插入突变体库,筛选出7株对3种病原细菌抑制效果明显下降的突变体,但其对立枯丝核菌的抑制效果与野生型菌株YL-1相同;PCR和Southern Blot试验结果表明绿针假单胞菌YL-1的7株突变体均是单拷贝;克隆到7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其中6个突变位点是嗜铁素合成基因簇,1个突变位点是蛋白分泌基因sec Y。

Cloning the genes of Pseudomonas chlororaphis YL-1 dedicated to antibacterial activities against microbial phytopathogens

Strain YL-1 was isolated from soybean root tips and identified to be Pseudomonas chlororaphis. This strain showed broad-spectrum antibacterial and antifungal activities against plant pathogens that are economically important in agriculture. In order to characterize the genes dedicated to antibacterial activities against microbial phytopathogens, a Tn5-mutation library of YL-1 was constructed. Plate bioassays revealed that seven mutants MT1-7 reduced their antibacterial activities against Burkholderia glumae, Erwinia amylovora and Pectobacterium carotovora as compared with its wild type strain while remained steady antifungal activity against Rhizoctonia solani. All the mutants MT1-7 were verified by PCR and Southern Blot, and the transposon was inserted into YL-1 genomic DNA successfully by single copy. Seven genes around the insertion site were cloned and sequenced using a plasmid rescue procedure as recommended by the manufacturer of the EZ-Tn5 kit. The bioinformatic analysis results showed that six genes were related to pyoverdine synthesis and the other secY gene belonged to the Sec protein translocation system.