西瓜细菌性果斑病菌鞭毛基因fliS的功能分析

发布时间：2018-12-12 19:36

【摘要】：【目的】西瓜细菌性果斑病由西瓜噬酸菌(Acidovorax citrulli,Ac)引起,是一种严重的世界性病害。细菌的鞭毛通常被认为是细菌的运动器官,在细菌的侵染过程中也起重要作用,已有报道表明这种作用可受鞭毛蛋白基因fliS的调控,目前西瓜细菌性果斑病菌鞭毛蛋白基因fliS的功能及其调控机理尚不清楚,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。【方法】以果斑病菌野生型致病菌株1号基因组DNA为模板,设计一系列引物,PCR扩增敲除基因fliS的上下游片段,通过回收、酶切、连接、转化等步骤构建敲除载体和互补载体,然后采用三亲杂交法,根据同源重组的原理,构建fliS基因缺失突变菌株及其互补菌株,并对其鞭毛的形态特征、致病性、过敏反应、游动性、群体感应、菌膜、生长速率、菌落形态等生物学特性进行测定;进一步提取细菌总RNA,以谷氨酰胺合成酶基因glnA为参照来校正目标基因的表达量,采用实时荧光定量PCR(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flh D、fliE、fliC、flgK、flgM、fliD和fliA的表达量差异。【结果】通过抗性基因Gm的筛选和PCR验证,成功构建了果斑病菌鞭毛蛋白基因fliS缺失突变菌株1-fliS及其互补菌株1-fliShb,并对所得菌株的生物学特性和鞭毛进行观察,结果表明,与野生菌株相比,鞭毛蛋白基因缺失突变菌株的游动性、菌膜形成能力减弱,互补后游动性、菌膜形成能力基本恢复;缺失突变菌株对甜瓜、西瓜幼苗以及西瓜果实的致病性降低,互补后对西瓜、甜瓜幼苗及西瓜果实的致病力完全恢复。电镜测试显示,突变菌株鞭毛变短,长度约为野生菌株的1/3—1/4,互补后鞭毛合成能力基本恢复,鞭毛长度约为野生菌的4/5;光学显微镜下,可观察到在NA平板上的野生菌株菌落周围有明显的由细菌颤泳形成的特殊晕圈,而缺失突变菌株在NA平板上不能形成这种晕圈,互补后晕圈形成能力部分恢复;缺失突变菌株的生长速率比野生菌株慢,互补后生长速率没有恢复;野生菌株、突变菌株和互补菌株在过敏性反应和群体感应方面无差异。qRT-PCR分析结果显示,fliS基因缺失突变后,flh D表达量较野生菌株明显降低,fliE、fliC和flgK表达量较野生菌株明显升高,flgM和fliD表达量略微上升,fliA表达量基本不变;互补菌株中flhD、fliE和fliC表达量部分恢复,flgK、flgM和fliD表达量没有恢复,与突变菌株相同。【结论】鞭毛基因fliS对果斑病菌鞭毛丝的形成、游动性、菌膜形成能力、生长速率、菌落形态、致病性等均有调控作用。
[Abstract]:[objective] the bacterial fruit spot of watermelon is caused by Acidovorax citrulli,Ac, which is a serious worldwide disease. The flagella of bacteria is generally considered to be the motility organ of bacteria and plays an important role in bacterial infection. It has been reported that this action can be regulated by the flagellin gene fliS. At present, the function and regulation mechanism of flagellin gene fliS in bacterial fruit spot pathogen of watermelon are not clear. The purpose of this study was to investigate the role of the gene in the biological characteristics of flagella formation and pathogenicity. [methods] A series of primers were designed based on the genomic DNA of wild type pathogenic bacteria strain 1. The upstream and downstream fragments of knockout gene fliS were amplified by PCR. The knockout vector and complementary vector were constructed by recovery, enzyme digestion, ligation and transformation. The mutant strain of fliS gene deletion and its complementary strain were constructed, and the biological characteristics of flagella such as morphology, pathogenicity, hypersensitivity, swimming, colony induction, membrane, growth rate and colony morphology were determined. The total bacterial RNA, was further extracted to correct the expression of the target gene using glutamine synthase gene glnA as the reference, and real-time fluorescence quantitative PCR (qRT-PCR) was used to compare the wild strains. Partial flagellin gene of knockout strain and complementary strain were differentially expressed between the two strains. [results] by screening the resistant gene Gm and verifying by PCR, the expression levels of fliD and fliA were different between the knockout strain and the complementary strain. The mutant strain 1-fliS with flagellin gene fliS deletion and its complementary strain 1-fliShbwere successfully constructed. The biological characteristics and flagella of the strain were observed. The flagellin gene deletion mutant strain had the ability of swimming, the ability of membrane formation was weakened, and the ability of membrane formation was basically recovered after complementation. The pathogenicity of the mutant to melon, watermelon seedling and watermelon fruit was decreased, and the pathogenicity of the mutant strain to watermelon, melon seedling and watermelon fruit was completely recovered after complementation. The results of electron microscopy showed that the flagella of the mutant strain became shorter and the length was about 1 / 3 / 1 / 4 of that of the wild strain, and the flagellum synthesis ability of the mutant was almost recovered after complementation, and the length of the flagella was about 4 / 5 of that of the wild strain. Under the optical microscope, it was observed that there was a special halo circle formed by bacterial fibrillation around the colony of wild strains on the NA plate, but the absent mutant strain could not form the halo circle on the NA plate, and the ability of halo formation was partly recovered after complementation. The growth rate of deletion mutant strain was slower than that of wild strain, but the growth rate did not recover after complementation. There was no difference in allergy and population induction between wild strain, mutant strain and complementary strain. QRT-PCR analysis showed that the expression of, flh D was significantly lower than that of wild strain after fliS gene deletion mutation, and fliE, was significantly lower than that of wild strain. The expression of fliC and flgK was significantly higher than that of wild strain, the expression of flgM and fliD was slightly increased, and the expression of fliA was almost unchanged. The expression of flhD,fliE and fliC was partially recovered in complementary strains, but the expression of flgK,flgM and fliD was not recovered, which was the same as that of mutant strains. [conclusion] flagellum gene fliS can induce flagellum formation, swimming, membrane formation and growth rate. Colony morphology, pathogenicity and so on all have the regulation function.
【作者单位】：福建农林大学植物保护学院;
【基金】：国家公益性行业(农业)科研专项(201003066-2) 福建省自然科学基金(2014J01084) 福建农林大学发展基金(KF2015058);福建农林大学科技创新专项基金(CXZX2016133)
【分类号】：S436.5

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