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1、作物學(xué)報(bào)ACTAAGRONOMICASINICA2011,37(6):1012?1019http://www.chinacrops.org/zwxb/ISSN0496-3490;CODENTSHPA9E-mail:xbzw@chinajournal.net.cnDOI:10.3724/SP.J.1006.2011.01012陸地棉耐鹽相關(guān)基因GhSAMS的克隆及表達(dá)*周凱宋麗艷葉武威王俊娟王德龍樊保香中國農(nóng)業(yè)科學(xué)院棉花研究所/棉花生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室/農(nóng)業(yè)部棉花遺傳改良重點(diǎn)實(shí)驗(yàn)室,河南安陽455000摘要:為了挖掘棉花耐鹽相關(guān)基因,根據(jù)陸
2、地棉耐鹽性抑制消減文庫中的一個(gè)S-腺苷甲硫氨酸合成酶基因的同源EST設(shè)計(jì)引物,利用RACE結(jié)合RT-PCR技術(shù)克隆陸地棉S-腺苷甲硫氨酸合成酶基因的cDNA全長,命名為GhSAMS。該cDNA全長1576bp,ORF為1182bp,編碼393個(gè)氨基酸的多肽。生物信息學(xué)分析表明GhSAMS蛋白與擬南芥、鹽地堿蓬、水稻中該蛋白的相似性分別為91%、93%和93%。系統(tǒng)發(fā)育樹結(jié)果顯示GhSAMS與鹽地堿蓬中該蛋白的親緣關(guān)系最近。Real-timePCR分析結(jié)果表明,GhSAMS的表達(dá)受鹽脅迫誘導(dǎo),在鹽敏感材料中誘導(dǎo)被推遲,而且,該基因表達(dá)水平在
3、耐鹽材料中9835中明顯高于在鹽敏感材料中S9612中。構(gòu)建了原核表達(dá)載體pET28-GhSAMS,經(jīng)IPTG誘導(dǎo),實(shí)現(xiàn)了GhSAMS在大腸桿菌中的表達(dá),為進(jìn)一步開展GhSAMS的遺傳轉(zhuǎn)化工作奠定了有益基礎(chǔ)。關(guān)鍵詞:陸地棉;鹽脅迫;S-腺苷甲硫氨酸合成酶基因;原核表達(dá)CloningandExpressionofGhSAMSGeneRelatedtoSalt-toleranceinGos-sypiumhirsutumL.*ZHOUKai,SONGLi-Yan,YEWu-Wei,WANGJun-Juan,WANGDe-Long,andFANB
4、ao-XiangCottonResearchInstitute,ChineseAcademyofAgriculturalScience/StateKeyLaboratoryofCottonBiology/KeyLaboratoryofCottonGeneticImprovement,MinistryofAgriculture,Anyang455000,ChinaAbstract:Asoneofmainabioticstressesinnature,saltstressdoesgreatharmtoplants,andseriouslyaf
5、fectplantgrowthanddevelopment.Simultaneously,thecropscultivatedinthesalinelandundergoawiderangeofyielddecline.Toexcavatesalt-tolerancegene,weclonedthecDNAofS-adenosyl-L-methioninesynthetasegenefromGossypiumhirsutumbyRACEandRT-PCR,whichwasnamedGhSAMS,withthecDNAfulllengtho
6、f1576bp,ORFof1182bp,andcoding393aminoacidresi-dues.BioinformaticsanalysisshowedGhSAMShasthesimilarityof91%,93%,and93%withArabidopsisthaliana,Suaedasalsa,andOryzasativa,respectively.PhylogeneticanalysisshowedGhSAMSwastheclosesttoSuaedasalsa,andReal-timePCRsuggestedthatGhSA
7、MSwasinducedbysaltstress,whiletheinductionwaspostponedinsaltsensitivitymaterial.ItshowedlowergeneexpressionlevelonsaltsensitivematerialZhongS9612relativetosaltresistancematerialZhong9835.Atthesametime,weestablishedprotokaryoticexpressionvectorpET28-GhSAMSandtransformedGhS
8、AMSintoE.coliafterIPTGinduction,showingasuccessfulgeneexpression.Keywords:Gossypiumhirsutum;Salt
