水稻基腐病菌flhDC和fliA基因的功能

Functional Analysis of flhDC and fliA in Dickeya zeae

【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、flh DC和fli A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flh DC和fli A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flh DC和fli A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flh DC和fli A的融合片段,双酶切纯化后连接到自杀性载体p KNG101上,构建带有反向筛选标记基因sac B的自杀重组质粒p KNG-Δflh DC和p KNG-ΔfliA,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flh DC和fli A缺失突变体Δflh DC和ΔfliA;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16Sr DNA为内参来校正目标基因的表达量,采用实时荧光定量PCR(q RT-PCR)方法,比较野生菌和突变体Δflh DC和ΔfliA下游基因flh D、flh C、fli A和fli C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Δflh DC和ΔfliA。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Δflh DC和ΔfliA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Δflh DC和ΔfliA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Δflh DC和ΔfliA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Δflh DC和ΔfliA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Δflh DC和ΔfliA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。q RT-PCR分析结果显示,在突变体菌株Δflh DC中,flh D和flh C不表达,fli A和fli C的表达量较野生菌明显下降;而在突变体ΔfliA中,flh D、flh C和fli A均不表达,fli C表达明显下降。【结论】调控细菌鞭毛基因表达的主调控因子flh DC操纵子,以及表达鞭毛特异性因子σ^(28)基因fli A,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,flh DC and fli A and their regulatory mechanisms are 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 flh DC and fli A in D.zeae.【Method】A set of primers were designed based on the genomic DNA of wild strain EC1 of D.zeae.The upstream and downstream fragments of target genes flh DC and fli A 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 of flh DC and fli A were obtained by PCR.After dualenzyme digestion and purification,the fusion fragments were connected to the suicide vector p KNG101,suicide recombinant plasmids p KNG-Δflh DC and p KNG-ΔfliA with reverse selection marker gene sac B were constructed,then transferred into wild strain EC1,respectively,by tri-parental mating,so the gene deletion mutants Δflh DC and ΔfliA 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（q RT-PCR） was carried out using 16 Sr DNA as internal control for normalization.Then the expression of downstream genes flh D,flh C,fli A and fli C in Δflh DC and ΔfliA was compared.【Result】Two target gene deletion mutants Δflh DC and ΔfliA 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 Δflh DC and ΔfliA were decreased obviously.The wild strain EC1 had a strong inhibitory effect on rice seed germination,while Δflh DC and ΔfliA significantly reduced the inhibition of rice 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 Δflh DC and ΔfliA inoculation only showed water-brown lesions around the inoculated sites.It indicated that Δflh DC and ΔfliA 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 q RT-PCR showed that in the mutant Δflh DC,the flh DC and the fli A did not express,while the expressions of the fli A and the fli C decreased obviously compared with the wild strain;In addition,the flh D,flh C and the fli A in the mutant ΔfliA did not express,but the expressions of the fli C decreased obviously.【Conclusion】The flh DC operon,which regulates the expression of the bacterial flagellum genes,and the fli A,which expresses flagellin specific factor σ^（28）,are important components of the bacterial flagellar system gene cluster.The genes flh DC and fli A significantly affect the motility,biofilm and the germination of rice seeds,and play an important role involving in the virulence in D.zeae.