绿针假单胞菌YL-1抗细菌活性相关基因的克隆和分析

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

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。

Strain YL-1 w as isolated from soybean root tips and identified to be Pseudomonas chlororaphis. This strain show ed 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 w as constructed. Plate bioassays revealed that seven mutants M T1-7 reduced their antibacterial activities against Burkholderia glumae,Erwinia amylovora and Pectobacterium carotovora as compared w ith its w ild type strain w hile remained steady antifungal activity against Rhizoctonia solani. All the mutants M T1-7 w ere verified by PCR and Southern Blot,and the transposon w as inserted into YL-1 genomic DNA successfully by single copy. Seven genes around the insertion site w ere cloned and sequenced using a plasmid rescue procedure as recommended by the manufacturer of the EZ-Tn5 kit. The bioinformatic analysis results show ed that six genes w ere related to pyoverdine synthesis and the other sec Y gene belonged to the Sec protein translocation system.