假结核耶尔森氏菌T6SS效应蛋白TepC功能研究  

Insecticidal activity of the transkingdom T6SS effector TepC in Yersinia pseudotuberculosis

作　　者：瞿熠晨 郭呈昊 赵依昕 史圆 邢照岩 宋莉 潘君风[1] 沈锡辉[1] QU Yichen;GUO Chenghao;ZHAO Yixin;SHI Yuan;XING Zhaoyan;SONG Li;PAN Junfeng;SHEN Xihui(College of Life Sciences,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100

出　　处：《西北农林科技大学学报(自然科学版)》2025年第5期47-55,共9页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金重点项目“跨界群体感应介导的细菌-真菌识别与响应机制”(32330004)。

摘　　要：【目的】研究模式病原细菌假结核耶尔森氏菌(Yersinia pseudotuberculosis)Ⅵ型分泌系统T6SS效应蛋白TepC对细菌生物膜形成的影响及其自身杀虫活性,为该效应蛋白的开发应用奠定基础。【方法】采用结晶紫染色法,观察假结核耶尔森氏菌野生型(WT)、ΔtepC突变体和ΔtepC(tepC)回补菌株的非生物表面生物膜形成情况,并对其进行定量和定性分析;以线虫模型进行生物表面的生物膜形成试验,研究TepC对假结核耶尔森氏菌生物膜形成能力的影响;以大蜡螟为模型,研究假结核耶尔森氏菌野生型和突变体对大蜡螟致病性的影响;以大蜡螟和棉铃虫为模型,研究纯化TepC蛋白的毒性作用;对大蜡螟脂肪体进行组织切片HE染色,研究TepC处理对大蜡螟脂肪体的影响;利用定量PCR方法研究假结核耶尔森氏菌野生型和tepC突变体感染对大蜡螟抗菌肽基因表达的影响;构建tepC转基因拟南芥,检测转基因拟南芥对甜菜夜蛾幼虫的毒性。【结果】TepC在假结核耶尔森氏菌生物膜形成过程中发挥重要作用,基因tepC的缺失导致假结核耶尔森氏菌在非生物表面和生物表面(线虫)的生物膜形成能力显著降低;TepC能影响假结核耶尔森氏菌对大蜡螟的致病性,注射假结核耶尔森氏菌野生型的大蜡螟存活率仅为36.7%,而注射ΔtepC突变体的大蜡螟存活率为70.0%,另外TepC蛋白对大蜡螟和棉铃虫均有毒性作用,具有广谱杀虫性;TepC处理导致大蜡螟脂肪体遭到破坏,其通过影响大蜡螟脂肪体发挥毒性作用;与接种假结核耶尔森氏菌ΔtepC突变体菌株相比,接种假结核耶尔森氏菌WT菌株的大蜡螟抗菌肽在6 h显著上调,表明TepC影响了大蜡螟的免疫防御功能,喂食tepC转基因拟南芥可以显著降低甜菜夜蛾幼虫的存活率。【结论】假结核耶尔森氏菌T6SS效应蛋白TepC不仅在细菌竞争中发挥作用,还具有杀虫功能,并能影响细菌生物膜的形成。【Objective】This research aimed to study the effect of T6SS effector TepC of the model pathogenic bacterium Yersinia pseudotuberculosis on biofilm formation and its own insecticidal activity,to lay a theoretical foundation for the development and application of this effector protein.【Method】Experiments were conducted to assess the effect of TepC on biofilm formation in Yersinia pseudotuberculosis with both abiotic surfaces and nematode models as biotic surfaces,crystal violet staining method was used to observe the biofilm formation of Yersinia pseudotuberculosis wild type(WT),ΔtepC mutant andΔtepC(tepC)complement strain,and a quantitative and qualitative analysis was conducted on the biofilm formation.The pathogenicity of Yersinia pseudotuberculosis wild-type andΔtepC mutant was studied with Galleria mellonella larvae as the model.Purified TepC protein was tested for its toxic effects on larvae of Galleria mellonella and Helicoverpa armigera.Additionally,HE staining on the fat body of Galleria mellonella larvae was conducted to investigate the toxic mechanism of TepC.Effects of Yersinia pseudotuberculosis wild-type andΔtepC mutant on the expression of antimicrobial peptide genes in Galleria mellonella larvae were studied with quantitative PCR.Furthermore,tepC transgenic Arabidopsis was constructed and its toxicity to Spodoptera exigua larvae was assessed.【Result】TepC played an important role in the biofilm formation of Yersinia pseudotuberculosis.Deleting the gene tepC greatly diminished the capacity of Yersinia pseudotuberculosis to produce biofilms on both abiotic surfaces(PVC)and biotic surfaces(nematodes).TepC had the potential to influence the pathogenicity of Yersinia pseudotuberculosis in Galleria mellonella.The survival rate of Galleria mellonella larvae injected with wild-type Yersinia pseudotuberculosis was just 36.7%,but the survival rate of Galleria mellonella injected with theΔtepC mutant was 70.0%.Furthermore,the TepC protein had toxicological impacts on both Galleria mellonella and

关 键 词：假结核耶尔森氏菌 Ⅵ型分泌系统 杀虫蛋白 生物膜 

分 类 号：S482.3[农业科学—农药学]