[目的]水稻细菌性基腐病(致病菌Dickeya zeae)是水稻重要细菌病害之一.细菌的鞭毛是重要的运动器官,迄今有关水稻基腐病菌的鞭毛系统、flhDC和fliA基因功能及其调控机理尚不清楚,明确这些鞭毛基因的功能,有利于进一步了解D.zeae的致病性综合调控网络、开发新型药物作用靶标以及制定病害防控策略.论文旨在明确D.zeae鞭毛系统中flhDC和fliA在致病性中的作用.[方法]以D.zeae野生型致病菌株EC1基因组DNA为模板,设计一系列引物,PCR扩增待敲除的目标基因fJhDC和fliA各自的上、下游片段,再以混合的上、下游片段为模板,扩增得到缺失flhDC和fliA的融合片段,双酶切纯化后连接到自杀性载体pKNG101上,构建带有反向筛选标记基因sacB的自杀重组质粒pKNG-△lhDC和pKNG-△fliA,通过三亲转化方法分别将重组质粒导入野生型菌株EC1中,通过两次等位基因同源重组,PCR检测和测序验证,最终获得目标基因flhDC和fliA缺失突变体△flhDC和△fliA;测定并比较突变体与野生菌的胞外酶活性、毒素活性、运动性、生物膜形成能力,以及对水稻的致病力和对烟草的过敏性反应(HR);进一步提取细菌总RNA,以16SrDNA为内参来校正目标基因的表达量,采用实时荧光定量PCR (qRT-PCR)方法,比较野生菌和突变体△flhDC和△fliA下游基因flhD、flhC、fliA和fliC的表达量差异.[结果]通过基因操作手段成功构建了基因缺失突变体△flhDC和△fliA.表型测定结果显示,野生菌EC1的运动性和形成生物膜的能力很强,而基因缺失菌株△flhDC和△fliA的运动性和形成生物膜的能力明显下降;野生菌株EC1对水稻种子萌发具有很强的抑制作用,而突变体△flhDC和△fliA则显著降低了对水稻种子萌发的抑制作用;接种野生菌株EC1的水稻植株产生大面积褐色病斑,且腐烂程度严重,而接种突变体△flhDC和△fliA的水稻植株只在接种的针刺部位周围出现水渍状褐色病斑,腐烂程度轻微,说明突变体菌株△flhDC和△fliA显著降低了对水稻植株上的致病力.进一步的表型测定结果显示,突变体菌株△flhDC和△fliA与野生菌EC1在产生胞外致病酶、毒素以及引起烟草HR能力等方面没有显著差异.qRT-PCR分析结果显示,在突变体菌株△flhDC中,flhD和lhC不表达,fliA和fliC的表达量较野生菌明显下降;而在突变体△fliA中,flhD、flhC和fliA均不表达,fliC表达明显下降.[结论]调控细菌鞭毛基因表达的主调控因子flhDC操纵子,以及表达鞭毛特异性因子σ28基因fliA,是细菌鞭毛系统基因簇的重要组分.flhDC和fliA显著影响D.Zeae的运动性和生物膜形成能力,并显著影响水稻种子的萌发功能,在水稻基腐病菌的致病性中起重要作用.%[Objective] Rice foot rot,caused by Dickeya zeae,is one of the important bacterial diseases on rice.Bacterial flagella is an important movement organ,so far,the mechanism of the flagellar system,flhDC and fliA and their regulatory mechanisms arc unclear in D.zeae.To clarify the function of these flagellum genes is helpful for further understanding the pathogenicity of integrated control network in D.zeae,developing new drug action targets and making disease prevention and control strategies.The objective of this study is to investigate the function of flagellar system of flhDC and fliA in D.zeae.[Method] A set of primers were designed based on the genomic DNA of wild strain EC1 ofD.zeae.The upstream and downstream fragments of target genes flhDC and fliA to be knocked out were amplified by PCR,respectively.The upstream and downstream fragments were mixed as a template,and then the fusion fragments that lack offlhDC andfliA were obtained by PCR.After dual enzyme digestion and purification,the fusion fragments were connected to the suicide vector pKNG101,suicide recombinant plasmids pKNG-△flhDC and pKNG-△fliA with reverse selection marker gene sacB were constructed,then transferred into wild strain EC1,respectively,by tri-parental mating,so the gene deletion mutants △flhDC and △fliA were constructed after two alleles homologous recombination screening and PCR detection and sequencing verification.The biological characteristics such as extracellular enzyme,toxin,motility,biofilm,virulence to rice and HR on tobacco were compared and analyzed.In addition,bacterial total RNA was extracted,and a real-time quantitative PCR (qRT-PCR) was carried out using 16SrDNA as internal control for normalization.Then the expression of downstream genesflhD,flhC,fliA andfliC in △flhDC and △fliA was compared.[Result] Two target gene deletion mutants △flhDC and △fliA were constructed successfully by genetic manipulation.Phenotypic test results showed that the motility and biofilm formation of wild strain EC1 were very strong,while the motility and biofilm formation of the △flhDC and △fliA were decreased obviously.The wild strain EC1 had a strong inhibitory effect on rice seed germination,while △flhDC and △fliA significantly reduced the inhibition office seed germination.The rice plants inoculated with the wild strain EC1 showed a brown spot and a large extent of rottenness,while rice plants with △flhDC and △fliA inoculation only showed water-brown lesions around the inoculated sites.It indicated that △flhDC and △fliA significantly reduced the virulence to rice plant.Further phenotypic results showed that the activities of extracellular enzymes,toxin and the ability to cause HR on tobacco were not different significantly between the mutants and the wild strain.The results of qRT-PCR showed that in the mutant △flhDC,the flhDC and the fliA did not express,while the expressions of the fliA and the fliC decreased obviously compared with the wild strain;In addition,the flhD,flhC and the fliA in the mutant △fliA did not express,but the expressions of the fliC decreased obviously.[Conclusion] TheflhDC operon,which regulates the expression of the bacterial flagellum genes,and thefliA,which expresses flagellin specific factor σ28,are important components of the bacterial flagellar system gene cluster.The genesflhDC and fliA significantly affect the motility,biofilm and the germination of rice seeds,and play an important role involving in the virulence in D.zeae.
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