陆地棉耐盐相关基因ghsams地克隆及表达

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1、作物学报ACTAAGRONOMICASINICA2011,37(6):10121019http://www.chinacrops.org/zwxb/ISSN0496-3490;CODENTSHPA9E-mail:xbzw@chinajournal.net.cnDOI:10.3724/SP.J.1006.2011.01012陆地棉耐盐相关基因GhSAMS的克隆及表达*周凯宋丽艳叶武威王俊娟王德龙樊保香中国农业科学院棉花研究所/棉花生物学国家重点实验室/农业部棉花遗传改良重点实验室,河南安阳455000摘要:为了挖掘棉花耐盐相关基因,根据陆

2、地棉耐盐性抑制消减文库中的一个S-腺苷甲硫氨酸合成酶基因的同源EST设计引物,利用RACE结合RT-PCR技术克隆陆地棉S-腺苷甲硫氨酸合成酶基因的cDNA全长,命名为GhSAMS。该cDNA全长1576bp,ORF为1182bp,编码393个氨基酸的多肽。生物信息学分析表明GhSAMS蛋白与拟南芥、盐地碱蓬、水稻中该蛋白的相似性分别为91%、93%和93%。系统发育树结果显示GhSAMS与盐地碱蓬中该蛋白的亲缘关系最近。Real-timePCR分析结果表明,GhSAMS的表达受盐胁迫诱导,在盐敏感材料中诱导被推迟,而且,该基因表达水平在

3、耐盐材料中9835中明显高于在盐敏感材料中S9612中。构建了原核表达载体pET28-GhSAMS,经IPTG诱导,实现了GhSAMS在大肠杆菌中的表达,为进一步开展GhSAMS的遗传转化工作奠定了有益基础。关键词:陆地棉;盐胁迫;S-腺苷甲硫氨酸合成酶基因;原核表达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