西瓜细菌性果斑病菌鞭毛基因fliS的功能分析

【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。

Function Analysis of Flagellar Gene fliS in Acidovorax citrulli

【Objective】 Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is one of the most serious diseases in the world. Flagella are always considered as movement organ of bacteria and play an important role in their infection, and they have been reported that they could be controlled by flagellar protein gene fliS. The function of gene fliS in A. citrulli is still unclear. The objective of this study is to investigate the function of gene fliS of A. citrulli ineffecting on flagellum formation and pathogenicity. 【Method】 A set of primes were designed based on the genomic DNA No.1 of A. citrulli wild strain. The upstream and downstream fragments knocked out gene fliS were amplified by PCR, respectively. The gene knockout and complementary vectors were constructed through PCR amplification, recovery, digestion, connection and transformation. The strains with knockout gene fliS and complementation were constructed with the method of triparental hybridization. Morphological characteristics of flagella, pathogenicity, hypersensitive response, motility, quorum sensing, biofilm formation, growth rate, colonial morphology, etc have been tested among the wild type, the mutant and the complementary strains. In addition, the total RNA of the bacteria was extracted, and a real-time quantitative PCR (qRT-PCR) was carried out using glnA as an internal control for normalization. Then the expression of genes, flhD, fliE, fliC, flgK, flgM, fliD and fliA in the wild type, the mutant and the complement strains were compared.【Result】The deletion mutant and complementary strain were obtained successfully by screening of gentamicin resistance and PCR verifying, named as mutant 1-fliS and complementary strain 1-fliShb. The results showed that the deletion mutant had weakened motility and biofilm formation, and the complementary strain was almost recovered. Compared with the wild strain, the pathogenicity of the mutant on watermelon and melon was reduced and the complementary strain was recovered completely. The length of flagellum of the deletion mutant was 1/3-1/4 of that of the wild strain, and the complementary strain almost recovered and it was about 4/5 of that of the wild strain. The wild strain could form typical haloes obviously by bacteria migrating via twitching on NA medium, but the deletion mutant did not form haloes on NA medium and the complementary strain recovered partially. The growth rate of deletion mutant was slower than the wild strain and the complementary strain was not recovered. There were no difference among the wild, mutant and complement strains on hypersensitive response and quorum sensing. The results of qRT-PCR showed that in the mutant the expressions of gene flhD decreased obviously compared with the wild strain. In addition, the expressions of fliE, fliC and flgK increased obviously, the expressions of flgM and fliD increased slightly, the expressions of fliA was constant compared with the wild strain, the expressions of genes flhD, fliE and fliC recovered partially in complementary strain and, the expressions of flgK, flgM and fliD did not recover. 【Conclusion】 The flagellar gene fliS could regulate the flagellum formation, motility, biofilm formation, growth rate, colony morphology and pathogenicity of A. citrulli.