甜瓜细菌性果斑病菌致病性突变体筛选与hrcR基因的克隆.pdfVIP

植物病理学报 ACTAPHYTOPATHOLOGICASINICA 39(5)：501。506(2009) 甜瓜细菌性果斑病菌致病性突变体 筛选与 hrcR基 因的克隆 任争光，侯 磊，宋治国，张力群 (中国农业大学植物病理学系农业部植物病理学重点开放实验室，北京 100193) 摘要：细菌性果斑病 (BacterialFruitBlotch，BFB)是西瓜和甜瓜的重要病害。本实验从发病甜瓜果实上分离得到致病菌 株MH21，经鉴定为燕麦食酸菌西瓜亚种 (Acidovoraxavenaesubsp．citrulli)。利用转座子Mini—Tn5构建MH21菌株的突变 体库，Southern印迹杂交结果显示Mini—Tn5在菌株MH21染色体上可随机、单拷贝插入。通过浸种处理甜瓜种子进行突变 体的致病性检查，筛选得到 1株致病性完全丧失的突变体M543。对 M543中转座子插入基因的克隆和测序表明其突变基 因为燕麦食酸菌Ⅲ型分泌系统(TTSS)中的保守基因hrcR。推测菌株MH21中hrcR基因编码的蛋 白定位于细菌内膜，是 分泌通道主要成分之一。hrcR基因互补菌株的致病性检查结果显示其致病力恢复到野生型的84％。研究同时发现hrcR 基因突变后MH21菌株丧失了在烟草上诱导过敏性坏死反应的能力。本研究从遗传学角度说明TTSS是西甜瓜果斑病菌 致病性的重要因子。 关键词：燕麦食酸菌；Tn5转座子；Ⅲ型分泌系统：hrcR基因 Screeningofthe path0genicity mutantsofAcidovorax avenae subsp．citrulliand cloningofthehrCR gene REN Zheng—guang，HOULei．SONGZhi。guo。ZHANGLi．qun The KeyLaboratoryofPlantPathology，M inistryofAgriculture，andDepartmentofPlantPathology，ChinaAgriculturalUniversity， Beijing100193，China) Abstract：TheBacterialFruitBlotch(BFB)causedbyAcidovoraxavenaesubsp．citrulliisaseriousdisease onwatermelonandmelon．A strainMH21wasisolatedfrom adiseasde melonfruit，andWas identifiedasA．ave— naesubsp．citrulli．Inthisstudy，thestrainMH21wasmutatedusingtransposonM ini—Tn5 toselectthemu— tantsdefectiveintheirpathogencityonmelon seedling．Southernblotanalysisrevealed thattransposon Tn5 generatedtherandom andsingle—copyinsertionsinhtechromosomeofstrainMH21．A mutnatM 543lostits pahtogenicityonmelonseedlingwasselectedandthegenedisruptedbyTn5 wasidentifiedtobethehomolog ofhrcRgene．whichcodedaconservedinner—membrnaeproteinofTypeIIIsecretionsystem (TTSS)insome wel1．documentedbacterialpathogens．ThepotexperimentsrevealedhtatpathogenicityofM543couldbecorn— plementedbyplasmid—born wide—typehrcR gene．Additionally，mutantM543defectiveinhrcR genealsolost itsabilitytotriggerhypersensitivereaction(HR)ontobaccoleaf．Thegeneticsevidenceinthisreportdemon stratedthathteTTSS iscriticalforthepathogenic