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1、植物遗传资源学报2010,11(5):605-610JournalofPlantGeneticResources小麦NBS类抗病基因同源cDNA序列的克隆与特征分析张楠,王海燕,刘大群(河北农业大学植物保护学院/河北省农作物病虫害生物防治工程技术研究中心,保定071001)摘要:根据已克隆植物抗病(R)基因NBS保守结构域设计简并引物,采用RT-PCR和cDNA末端快速扩增技术(RACE),在小麦抗叶锈病近等基因系材料TcLr19中进行抗病同源基因cDNA全长的扩增。获得了1个通读的NBS类抗病同源基因S11A11cDNA序列,该序列全长2923bp,编码878个氨基酸序列。生
2、物信息学分析结果表明,该片段含有NB-ARC保守结构域和多个LRR结构域。聚类分析表明,S11A11编码的蛋白与小麦抗叶锈病基因Lr1编码的蛋白亲缘关系较近,而与Lr10亲缘关系较远。半定量RT-PCR分析表明,该基因在小麦叶片中为低丰度组成型表达。本研究在TcLr19小麦中成功获得了抗病基因同源序列,为最终克隆小麦抗叶锈病目的基因奠定了基础。关键词:小麦;核苷酸结合位点(NBS);抗病基因同源序列(RGAs);cDNA末端快速扩增技术(RACE);半定量RT-PCRCloningandCharacterizationofaNBSResistanceGeneHomologycDNAS
3、equencefromWheatZHANGNan,WANGHa-iyan,LIUDa-qun(CollegeofPlantProtection,AgriculturalUniversityofHebei/BiologicalControlCenterofPlantDiseaseandPlantPestsofHebeiProvince,Baoding071001)Abstract:Resistancegenehomologysequencefromwheatwasisolatedbyusinghomology-basedmethod.Apairofdegeneratedprimer
4、wasdesignedaccordingtothenucleotidebindingsiteconserveddomainsoftheclonedplantdiseaseresistance(R)genes.RT-PCRandRACEwereusedtoobtainthefulllengthsequenceofthediseaseresistancehomologygeneinthenearisogeniclinesTcLr19.Oneopen-readingNBSclassofresistancegeneanalogs(RGAs)namedS11A11wasobtained,whi
5、chwas2923bpinlengthandencoded878aminoacids.Bioinformaticsa-nalysisshowedthededucedaminoacidsofS11A11proteinconsistedofaNB-ARCconserveddomainandmanyleucine-richrepeats(LRR)domains.ThephylogenetictreeanalysisindicatedaconsiderableidentityoftheproteinencodedbyS11A11withthatofwheatleafrustresistanc
6、egeneLr1,butalowersimilaritywithLr10.TheS11A11geneappearednottobeinducedbyPucciniatriticinaandwasaconstitutivegenewithlowabundanceinwheatleaftissuebysem-iquantitativeRT-PCR.TheresistancehomologysequencewassuccessfullyobtainedinTcLr19,whichprovidestheshortcutforcloningofwheatleafrustresistancege
7、ne.Keywords:Whea;tNucleotidebindingsite(NBS);Resistancegeneanalogs(RGAs);RapidamplificationcDNAend(RACE);Sem-iquantitativeRT-PCR小麦(TriticumaestivumL.)适应性强,分布广,是产的重要病害之一,也是影响我国小麦生产的重要因世界上最重要的粮食作物,由小麦叶锈菌(Puccinia素,其分布范围比条锈病和秆锈病更广,
