西瓜噬酸菌Ⅳ型菌毛相关基因pilO及pilQ的功能分析  

Functional Analysis of Type Ⅳ Pili Related Genes pilO and pilQ in Acidovorax citrulli

作　　者：王敏鑫[1] 武珍[1] 邱晓静[1] 王健超[1] 田艳丽[1] 胡白石[1,2] 刘凤权[1] 

机构地区：[1]南京农业大学植物保护学院农作物生物灾害综合治理教育部重点实验室,南京210095 [2]国家瓜类工程技术研究中心,昌吉831100

出　　处：《农业生物技术学报》2013年第6期716-724,共9页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展计划项目(No.2009CB119200);全国科技支疆计划项目(No.201191133);公益性行业(农业)科研专项(No.201003066)

摘　　要：瓜类细菌性果斑病是发生在葫芦科植物上的一种病害,其病原为噬酸菌属西瓜种(Acidovorax citrulli),自报道以来已给美国、澳大利亚、巴西、中国等众多国家的瓜类作物种植业造成了严重的损失。要从根本上防治瓜类细菌性果斑病,我们应弄清其致病机制。本实验对西瓜噬酸菌(A.citrulli)中部分Ⅳ型菌毛(pili)相关基因构建插入突变体,用浸种测定法筛选出两株致病力和发病率明显减弱的突变体,鉴定为pilO及pilQ基因突变体,并对这两株突变体的相关表型进行测定。在透射电子显微镜下观测发现,野生型及pilO基因突变体仍然能够形成Ⅳ型菌毛,而pilQ基因突变体丧失形成Ⅳ型菌毛的能力;光学显微镜下观测野生型、突变体及互补菌株的颤动能力发现,pilO及pilQ基因突变体与野生型相比,完全丧失颤动能力;同时pilO及pilQ基因突变体游动能力、生物膜形成能力也明显减弱。互补菌株中,颤动能力得到恢复,致病力、游动能力以及生物膜形成能力部分恢复。在MMX基本培养基中测定野生型与突变体的生长曲线发现,突变体菌株生长能力无明显改变;同时突变体也能激发非寄主植物烟草(Nicotiana tabacum)的过敏性反应。pilO基因突变体仍然能够形成Ⅳ型菌毛但丧失部分Ⅳ型菌毛介导的功能,由此表明,pilO基因并不直接参与西瓜噬酸菌Ⅳ型菌毛的合成,而是Ⅳ型菌毛的功能基因;pilQ基因突变体不能形成Ⅳ型菌毛且丧失其介导的部分功能表明,pilQ基因在西瓜噬酸菌中直接参与Ⅳ型菌毛的合成,是Ⅳ型菌毛的合成及功能基因。同时也进一步证明西瓜噬酸菌Ⅳ型菌毛与其颤动性、致病性、游动性及生物膜形成能力相关。Bacterial fruit blotch of melons is an important disease on cucurbit plants which is caused by Acidovorax citrulli. Since now it has been reported that this disease can cause serious damages of local melons in United States, Australia, Brazil, China and many other countries. So far, to preventbacterial fruit blotch of melons, we should find out the mechanism ofAcidovorax citrulli. In this experiment, we constructed several type Ⅳ pili related gene-inserted mutants, and obtained two mutants with dramaticallyreduced virulence by seed-transmission assays which was identified for pilO and pilQ genes, then we tested some related phenotypes about them. By transmission electron microscopy, we found that wild type and pilOmutant could form type Ⅳ pili, whereas pilQ mutant lost type Ⅳ pili. Results of optical microscope observation showed that pilO and pilQ mutants, compared with wild type, both lost twitching motilitycompletely, meanwhile their swimming ability and biofilm formation also reduced significantly. For complementary strains, virulence, twitching motility, swimming ability and biofilm formation recovered partly.Compared with wild-type, the growth of mutants showed no significant difference in MMX medium, and they could also stimulate hypersensitive response in non-host. PilO mutant lost some abilities mediated by type Ⅳpili but still could form type Ⅳ pili showed that, pilO gene was not directly involved in synthesis of type Ⅳ pill, but it was functional gene of type Ⅳ pill in A. citrulli. PilQ mutant not only lost the abilities mediated bytype Ⅳ pili but also couldn＇ t form type Ⅳ pili showed that, pilQ gene was directly involved in synthesis of type Ⅳ pili and was the synthetic and functional gene of type Ⅳ pili in A. citrulli. From this we knew that type Ⅳ pili ofA. citrulli was related to twitching motility, virulence, swimming ability and biofilm formation.

关 键 词：西瓜噬酸菌 Ⅳ型菌毛 颤动能力 致病性 

分 类 号：S651[农业科学—果树学]