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1、21卷6期生物工程学报Vol.21No.62005年11月ChineseJournalofBiotechnologyNovember2005烟草MARs的分离及其功能分析IsolationandFunctionalAnalysisofTobaccoMARs1,21*111黄慧珍,王瑶,陈士云,王志华,杨宝玉1,21*111HUANGHuiZhen,WANGYao,CHENShiYun,WANGZhiHuaandYANGBaoYu1中国科学院武汉病毒研究所,武汉4300712西北农林科技大学农学院,杨凌71
2、21001WuhanInstituteofVirology,ChineseAcademyofSciences,Wuhan430071,China2DepartmentofAgronomy,NorthwestSciTechUniversityofAgricultureandForestry,Yangling712100,China摘要从烟草基因组中克隆到两条新的MAR片段(M14和M17),序列分析表明,它们具有90%ATbox,Abox,Tbox,碱基非配对区域,拓扑异构酶识别位点,弯曲DNA序列,复制起始序列和ATAT
3、TT等典型的MAR序列特征,并与原有MAR序列的特征不同。将它们分别构建到植物表达载体pCAMBIA2301GUS基因(uidA)表达盒一侧及两侧,通过农杆菌介导转化烟草。组织化学染色法定性检测GUS活性表明,带有M14和M17的uidA基因在转基因烟草中稳定表达。GUS活性的定量检测表明,表达载体上uidA基因一端或两端连接有MAR的转化烟草中,GUS的表达水平与对照相比都有了明显提高,而uidA基因两侧连有MAR的载体提高表达水平的效果优于一端连有MAR的载体,可使GUS活性增强314倍,但不同转化个体之间表达水平的差异仍然明显
4、。上述结果表明,所得DNA序列为两条新的MAR片段,并且具有提高转基因表达水平的功能。关键词MARs,烟草,葡糖醛酸酶,转基因表达中图分类号Q78文献标识码A文章编号10003061(2005)06097005AbstractTwonewMARsegments(M14andM17)wereclonedfromtobaccogenome.BothofthesequencescontainedseveraltypicalconsensussequencesofMARs,whichweredifferentfrom
5、theoriginalMARsequence,suchas90%ATbox,Abox,Tbox,thebaseunpairingregions(BUR),autonomouslyreplicatingsequences(ARS),theconsensussequencefortopoisomerase,MARrecognitionsequence(MRS),originofreplication(ORI),curvedDNAmotifsandATATTTetal.Toinvestigatetheeffectsofthesetw
6、osequencesongeneexpressionintransgenicplants,3plantexpressionvectorswereconstructedwithuidAgenecodingglucuronidase(GUS)whichwereflankedononesideandonbothsidesbytheMARsweobtained.TheseplantexpressionvectorswithoneortwoMARsweretransformedintotobaccosviaAgrobacteriummed
7、iatedtransformationmethod,withtheplantexpressionvectorpCAMBIA2301withoutMARandwildtypetobaccoascontrols.GUShistochemicalstainingresultsshowedthattheuidAgeneexpressedstablyintransgenictobaccos.QuantitativedetectionofGUSactivityshowedthattheMARscouldincreaseGUSexpressionl
8、evelsinvivoincontrasttothecontrols,wherevertheywereflankedononesideorbothsidesofuidAgene.ThevectorligatedwithM
