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1、5期李會勇等:應(yīng)用抑制差減雜交法分離玉米幼苗期葉片土壤干旱誘導(dǎo)的基因887應(yīng)用抑制差減雜交法分離玉米幼苗期葉片土壤干旱誘導(dǎo)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(xué)院作物科學(xué)研究所,北京100081;2北京市農(nóng)林科學(xué)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(dǎo)表達(dá)的基因?!痉椒ā勘狙芯坷靡种撇顪p雜交(suppressionsubtractivehybridizatio
2、n,SSH)的方法,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?!窘Y(jié)果】在這個文庫中,隨機(jī)挑取了672個陽性克隆,并進(jìn)行PCR驗證,對所有的單克隆進(jìn)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
3、為未知功能蛋白和假定蛋白,4個uniESTs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(dǎo);抑制差減雜交法;耐旱基因IsolatingSoilDrought-inducedGenesfromMaizeSeedlingsLeavesThroughSuppressionSubtractiveHybridizationLIHui-yong1,HUANGSu-hua1,ZHAOJiu-ran2,WANGFeng-ge2,ZHANGZ
4、hong-bao1,MAOYi-hui1,WANGXiu-tang1,SHIYun-su1,SONGYan-chun1,WANGGuo-ying1,LIYu1,WANGTian-yu1(1InstituteofCropSciences,ChineseAcademyofAgriculturalSciences,Beijing100081;2MaizeResearchCenter,BeijingAcademyofAgriculturalandForestrySciences,Beijing100097)Abst
5、ract:【Objective】Isolationandanalysissoildrought-inducedgenesfrommaizeseedlingsleaves.【Method】AforwardcDNAlibrarywasconstructedbysuppressionsubtractivehybridizationusingseedlingleavesof“CN165”,adrought-tolerantmaizeinbredline.【Result】IntheSSHlibrary,672posi
6、tivecloneswerepickeduprandomly.AfterPCRofeachclone,allthesingleclonesweresequenced.Totally598availablesequenceswereobtained.AfterclusteranslysisoftheESTssequences,80uniESTswereobtained,amongwhich57uniESTswerecontigsand23uniESTsweresinglets.TheresultsofBLAS
7、TNshowedthatalltheuniESTshadhomologoussequencesinthenrdatabase.TheBLASTXresultsindicatedthat68uniESTshadsignificantproteinhomology,eightuniESTswereunknownproteinsandputativeproteins,andfouruniESTshadnoproteinhomology.【Conclusion】Alargegroupofdroughtstress-
8、inducedgeneswerefoundinthecDNAlibrary,whichinvolvedinmanymetabolismpathways.Keywords:Maizeseedlings;Droughtstress;Suppressionsubtractivehybridization(SSH);uniESTs5期李會勇等:應(yīng)用抑制差減雜交法分離玉米幼苗期葉片土壤干旱誘導(dǎo)的基因8870引言【研究意義】