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西瓜噬酸菌鞭毛基因flgM的功能分析

Function Analysis of Flagellin Gene flgM in Acidovorax citrulli
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摘要 西瓜(Citrullus lanatus)细菌性果斑病(bacterial fruit blotch, BFB)是世界范围内的检疫性病害,该病害的病原菌为西瓜噬酸菌(Acidovorax citrulli, Ac),自发现以来给西瓜种植业造成严重的经济损失,为了有效控制其危害,需要弄清该病菌的致病机制。细菌的鞭毛是细菌的运动器官之一,在细菌的侵染过程中也起着重要的作用。在多种不同细菌中已经证实,反σ因子flgM是调控鞭毛基因转录的必要因子,但是其在西瓜噬酸菌中的功能仍然未知,本研究旨在探讨该基因在鞭毛形成和致病性等生物学特性中的作用。根据同源重组的原理,借助自杀质粒p K8mobsacB,构建flgM基因缺失突变菌株,并对其鞭毛的形态特征、致病性、致敏性、游动性、群体感应、菌膜形成、生长速率及细菌颤泳性等生物学特性进行测定;以谷胱酰胺合成酶基因(glutathiamide synthetase gene, glnA)为参照基因,采用qRT-PCR方法,比较野生菌株、敲除菌株和互补菌株部分鞭毛蛋白基因flhD、fliE、fliC、flgK、flgA、fliS、fliD和fliA的表达量差异。结果表明,通过庆大霉素(gentamicin, Gm)抗性基因Gm筛选和PCR验证,成功地构建了西瓜噬酸菌鞭毛蛋白基因flgM的缺失突变菌株FJAc01-flgM及其互补菌株FJAc01-flgMhb,并对所得菌株的生物学特性进行观察,结果显示,与野生菌株相比,基因flgM缺失后不形成鞭毛、菌株的游动能力和菌膜形成能力均减弱、生长速率显著加快、对西瓜果实和西瓜苗的致病力明显减弱;基因互补后恢复了鞭毛的形成能力、游动性和菌膜形成能力、菌株的生长能力和对西瓜果实和西瓜苗的致病力也基本得到恢复,如针刺接种西瓜果实和喷雾接种西瓜苗后5 d,野生菌株处理的病情指数分别为74.67和26.39,基因缺失突变体处理的病情指数分别为46.00和2.78,而互补菌株处理的病情指数分别为63.85和20.83;光学显微镜下,可观察到在肉汁胨培养� Bacterial fruit blotch(BFB) caused by Acidovorax citrulli(Ac) is one of the most important destructive seed borne diseases on watermelon(Citrullus lanatus) and melon(Cucumis melo) in the world and the cause pathogen is a worldwide quarantine pest. This disease causes serious economic loss to watermelon production. In order to effectively control the disease, The pathogenicity mechanism of A. citrulli on watermelon is needed to know. Flagellum is a movement organ of bacteria and plays an important role in bacterial infection. Alterative anti-sigma factor flgM, required in the regulation of flagellar gene transcription,has been verified in different bacteria species, but its feature in A. citrulli is unclear. The objective of this study is to understand the function of gene flgM on flagellum formation and pathogenicity of A. citrulli. The flgM gene deletion mutant was generated by homologous recombination with the help of suicide plasmid pK8 mobsacB. Morphological characteristics have been tested of the flagella, pathogenicity, hypersensitive response, motility, quorum sensing, biofilm formation, growth rate, twitching, etc. among the wild type, the mutant and the complementary strain. Moreover, a real-time quantitative PCR(qRT-PCR) was carried out to compare the expression of genes, flhD, fliE, fliC, flgK, flgA, fliS, fliD and fliA in the wild type strain, the deletion mutant strain and the complementary strain using glutathiamide synthetase gene(glnA) as a reference.The results showed that the deletion mutant FJAc01-flgM and complementary strain FJAc01-flgMhb were generated successfully after gentamicin resistant screening and verified by PCR. Compared to wild type strain,the deletion mutant did not grow flagella and greatly attenuated in biofilm, motility, colonial morphology and the virulence on watermelon, but significantly accelerated in growth rate, which could be restored in the complementary strain. After 5 d of stabbed inoculation on watermelon fruit and spray inoculation on watermelon seedling, the disea
作者 杨丙烨 胡方平 蔡学清 YANG Bing-Ye;HU Fang-Ping;CAI Xue-Qing(State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops,College of Plant Protection,Fujian Agriculture and Forest University,Fuzhou 350002,China)
出处 《农业生物技术学报》 CAS CSCD 北大核心 2019年第3期504-515,共12页 Journal of Agricultural Biotechnology
基金 国家重点研发计划项目(No.2018YFD0201300) 福建农林大学科技创新专项基金(No.CXZX2016133).
关键词 西瓜噬酸菌 鞭毛 flgM基因 游动性 致病性 Acidovorax citrulli Flagella flgM gene Motility Pathogenicity
作者简介 通讯作者:蔡学清,caixq90@163.com
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