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

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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】