一株高拮抗活性菌株的鉴定及其抗菌活性缺失突变体的获得

Identification of A Highly Antagonistic Strain and Acquisition of Its Mutant Lack of Antimicrobial Activity

本研究以黄瓜枯萎病菌(Fusarium oxysporum)和青枯劳尔氏菌(Ralstonia solanacearum)为指示菌,从大豆植物根际土壤中筛选到一株拮抗活性较高的菌株(D-7),经Rec A基因序列分析,鉴定其为新洋葱伯克霍尔德氏菌(Burkholderia cenocepacia)。经PCR检测,该菌株不存在毒性基因BCESM(esmR)和cbl A。致病性分析发现该菌株对苜蓿幼苗和洋葱没有致病性。为了解该菌株的抗菌物质合成相关基因,利用Tn5转座子突变法构建菌株D-7的突变体库,并筛选获得10株对黄瓜枯萎病菌抗菌活性减弱的突变体。对各突变株插入位点分析发现,其中5个突变株的Tn5转座子插入到同一基因,该基因编码葡萄糖抑制的分裂蛋白A(Glucose-inhibited division protein A,Gid A),其余5株突变体的插入位点分别位于不同的基因上,编码的蛋白包括糖基转移酶(Glycosyl transferase)、Ⅱ型分泌系统蛋白(typeⅡsecretion system protein)等,表明菌株D-7的抗真菌能力有多个基因参与。

A bacteria strain D-7 was isolated from soybean rhizosphere soil and showed high anti-microbial activity to plant pathogenic fungi and bacteria,such as Fusarium oxysporum and Ralstonia solanacearum.The strain was identified to be Burkholderia cenocepacia based on RecA gene sequence and phylogenetic analysis.Specific PCR was constructed to amplify virulence genes,such as BCESM(esmR)and cblA,and the results showed the strain D-7 did not have the two genes.It also had no pathogenicity to alfalfa and onion.In order to understand the genes related to antibacterial substances of the strain D-7,a random mutant library was constructed using Tn5 transposon mutation method,and 10 mutants with reduced antimicrobial activity to Fusarium oxysporum were obtained.By the mutant insertion site analysis,we found that the Tn5 transposon in 5 mutants were inserted into the same gene,which encoded glucose-inhibited division protein A(GidA),while the insertion sites of the remaining 5 mutants were located on different genes,and the encoded proteins included glycosyl transferase,typeⅡsecretion system protein and so on.It indicated that there were multiple genes involved in the antifungal activity of strain D-7.