解淀粉芽胞杆菌SQR9亲缘识别与辨别相关基因的筛选

Screening of genes related to kin recognition and discrimination in Bacillus amyloliquefaciens SQR9

[目的]依据不同基因突变体的群集运动表型研究解淀粉芽胞杆菌SQR9中参与亲缘识别和辨别过程的关键基因。[方法]利用带有mariner转座元件的穿梭质粒pMarA,构建菌株SQR9的突变体文库,同时结合实验室已保存突变体,通过Swarming平板对峙表型进行筛选,找到与菌株SQR9和FZB42表型发生变化的突变株。针对Swarming表型变化转座子突变株,利用反筛标记pheS进行基因无痕敲除验证与表型验证。[结果]敲除菌株SQR9的sinR、fliD、flhO和degU基因后影响其亲缘识别野生型亲本菌株的能力。定点敲除菌株SQR9的flhO基因后发现,Swarming表型与Tn转座插入突变体一致,都与野生型菌株SQR9形成边界表型,可能参与亲缘识别过程。敲除spo 0A调控基因显著影响菌株SQR9的功能,其与解淀粉芽胞杆菌FZB42的对峙边界表型消失,可能参与非亲缘排斥。[结论]解淀粉芽胞杆菌SQR9中全局性调控基因(spo 0A、sinR和degU)、鞭毛蛋白合成相关基因(fliD和flhO)、芽孢萌发基因(gerAA和gerAB)、抗生素合成基因(srfAA)及其他基因(patB和yugK)可能参与菌株SQR9的亲缘识别与辨别系统。

[Objectives]We investigated the key genes involved in kin recognition and discrimination process of Bacillus amyloliquefaciens SQR9 by using different kinds of gene mutants.[Methods]A shuttle plasmid pMarA,which contained a mariner-based transposon,was used to construct a random insertion mutants library of B.amyloliquefaciens SQR9,and the mutants store in the laboratory were used as a supplemental source for the experiments.Screening of all mutants based on the swarming phenotype,when the swarming phenotype between mutants and SQR9/FZB42 were changed compared with phenotype of wild-type strain.If the target mutant was from transposon insertion mutants library,we confirmed its swarming phenotype by constructing the marker-free chromosome deletion mutant which using the counter-selectable marker pheS.[Results]Results showed that deletion of sinR,fliD,flhO and degU genes of strain SQR9 affected the ability to be recognized as kin by the wild-type parent.After knockout of the gene flhO of strain SQR9,the swarming phenotype was found to be consistent with the Tn transposition insertion mutant,and both formed boundary phenotype with the wild-type strain SQR9,indicating that gene flhO was probably involved in kin recognition of strain SQR9.Removal of regulation gene spo 0A had a strong effect on strain SQR9,and produced no boundary lines between mutantΔspo 0A and FZB42,possibly contributing to its designation as non-kin.[Conclusions]In B.amyloliquefaciens SQR9,global regulation genes(spo 0A,sinR and degU),flagellar protein synthesis genes(fliD and flhO),spore germination genes(gerAA and gerAB),antibiotic synthesis gene(srfAA)and other genes(patB and yugK)are potentially involved in kin recognition and discrimination system.