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1、5期李會勇等:應用抑制差減雜交法分離玉米幼苗期葉片土壤干旱誘導的基因887應用抑制差減雜交法分離玉米幼苗期葉片土壤干旱誘導的基因李會勇1,黃素華1,趙久然2,王鳳格2,張中保1,毛毅輝1,王秀堂1,石云素1,宋燕春1,王國英1,黎裕1,王天宇1(1中國農(nóng)業(yè)科學院作物科學研究所,北京100081;2北京市農(nóng)林科學院玉米研究中心,北京100097)摘要:【目的】分離土壤干旱脅迫條件下玉米幼苗期葉片誘導表達的基因?!痉椒ā勘狙芯坷靡种撇顪p雜交(suppressionsubtractivehybridization,SSH)的方法,
2、以耐旱自交系CN165為材料,構(gòu)建了土壤干旱脅迫下玉米幼苗葉片的正向抑制差減cDNA文庫。【結(jié)果】在這個文庫中,隨機挑取了672個陽性克隆,并進行PCR驗證,對所有的單克隆進行了測序。得到了598個有效序列,經(jīng)過EST聚類分析后,共得到了80個uniESTs。其中57個為contigs。23個為singlets。BLASTN的結(jié)果表明,所有的uniESTs都可以在玉米的核酸數(shù)據(jù)庫中找到同源序列。BLASTX的結(jié)果表明:68個uniESTs和已知功能的蛋白有高度的相似性,8個uniESTs為未知功能蛋白和假定蛋白,4個uniES
3、Ts沒有蛋白質(zhì)的相似性?!窘Y(jié)論】在這個cDNA文庫中發(fā)現(xiàn)了大量抗旱相關(guān)的基因,這些基因涉及到植物代謝的多種途徑。關(guān)鍵詞:玉米;干旱誘導;抑制差減雜交法;耐旱基因IsolatingSoilDrought-inducedGenesfromMaizeSeedlingsLeavesThroughSuppressionSubtractiveHybridizationLIHui-yong1,HUANGSu-hua1,ZHAOJiu-ran2,WANGFeng-ge2,ZHANGZhong-bao1,MAOYi-hui1,WANGXiu-t
4、ang1,SHIYun-su1,SONGYan-chun1,WANGGuo-ying1,LIYu1,WANGTian-yu1(1InstituteofCropSciences,ChineseAcademyofAgriculturalSciences,Beijing100081;2MaizeResearchCenter,BeijingAcademyofAgriculturalandForestrySciences,Beijing100097)Abstract:【Objective】Isolationandanalysissoil
5、drought-inducedgenesfrommaizeseedlingsleaves.【Method】AforwardcDNAlibrarywasconstructedbysuppressionsubtractivehybridizationusingseedlingleavesof“CN165”,adrought-tolerantmaizeinbredline.【Result】IntheSSHlibrary,672positivecloneswerepickeduprandomly.AfterPCRofeachclone
6、,allthesingleclonesweresequenced.Totally598availablesequenceswereobtained.AfterclusteranslysisoftheESTssequences,80uniESTswereobtained,amongwhich57uniESTswerecontigsand23uniESTsweresinglets.TheresultsofBLASTNshowedthatalltheuniESTshadhomologoussequencesinthenrdataba
7、se.TheBLASTXresultsindicatedthat68uniESTshadsignificantproteinhomology,eightuniESTswereunknownproteinsandputativeproteins,andfouruniESTshadnoproteinhomology.【Conclusion】Alargegroupofdroughtstress-inducedgeneswerefoundinthecDNAlibrary,whichinvolvedinmanymetabolismpat
8、hways.Keywords:Maizeseedlings;Droughtstress;Suppressionsubtractivehybridization(SSH);uniESTs5期李會勇等:應用抑制差減雜交法分離玉米幼苗期葉片土壤干旱誘導的基因8870引言【研究意義】