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ID:32011967
大小:1.61 MB
页数:54页
时间:2019-01-30
《番茄花粉高赖氨酸基因tsb导入中间偃麦草及转基因植株再生》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
1、内蒙古农业大学硕士学位论文番茄花粉高赖氨酸基因TSB导入中间偃麦草及转基因植株再生姓名:尹利申请学位级别:硕士专业:作物遗传育种指导教师:逯晓萍20070501摘要本实验通过RT_PcR从番茄花粉中获得高效特异表达的高赖氨酸基因TSB的cDNA序列,构建其受控于单子叶植物Ubil强启动子下的高效表达质粒pBIUB-TSB。与此同时,以禾本科牧草中间偃麦草为材料,以它的成熟种子为外植体,建立简单的植株再生体系,并将构建好的表达载体通过基因枪和激光微束穿刺转化法将其导入到中间偃麦草愈伤组织中,通过卡那霉素筛选,得到再生植株。实验结果表
2、明:我们建立了中间偃麦草简单的植株再生体系:(1)愈伤诱导培养基:MS+2.4.D(4.5mg/L)+BA0.1ing/L+蔗糖309几+琼脂6.5e,/LpH值5.8~6.O;(2)愈伤继代培养基:MS+2.4一D4mg/L+BAO.Img/L+Vc5mg/L+蔗糖309+琼脂6.59/L+$t醇100mg/LDH值5.8~6.0;(3)芽分化培养基:MS+BA2mg/L+NAAImg/L+CoCh5mg/L+ZTO,5mg门L+蔗糖309+肌醇100mg/L+琼脂6.59:LPH值5.8~6.O;(4)生根培养基:112MS+
3、IAA0.5mg/L+NAA0.5mg/L+活性炭0.1'针蔗糖308+琼脂6.59/Lpn值5.8~6.0。在卡那霉素为300mg/L的筛选压力下,两种转化法都得到了一些再生植株,经过PCR和Southernblotting检测,有4株成阳性,证明高赖氨酸基因TSB已经整合到了中间偃麦草的基因组中,其中通过基因枪转化获得2株,激光微束穿刺转化获得2株。关键词:中间偃麦草;高赖氨酸基因;转基因;组织培养GeneticTransformationofaPollenLysine—richGenefromTomatotoThinopyr
4、umintermediumandRegenerationofTransgenicPlantsAbstractThestudyinthispaper,aPollen-specificLysine—richProteineDNA(TSB),fromtomatobyRT-PCRwasobtained.AplantexpressionplasmidpBIUB—TSBwasconstructedandlysgenewasunderthecontrolofmaizeubiquitinpromoterwhichisthehighesteffic
5、ientmonocotyledonpromoter.Withthat,thetissecultureandplantregenerationwasconstructedusingthematureseedsofgramineousforage·Thinopyruraintermea&um.theplasmidpBn_rB—TSBwa$translateintothecallusoftheThinopyrumintermediumbythewayofMicroprojectilebombardmentandLaserMicrobea
6、mPunctureTechnique.RegeneratedfertiletransformedplantswereobtainedbyselectingforKanamyeinresistancethenpolarizatedthenewplanttranslation.Theexperimentalresultsshowedthat:Thecallusinducingmedium:(1)MS+2.4-D4.5mg/L+BA0.1mg/L+Sucrose30∥L+Agar6.59几pH:5.8~6.O;(2)Subculture
7、medium:MS+24-D4mg/L+BAO.Img/L+Ve5mg/L+Sucrose309/L+Aqgar6.59/L+InositollOOmg/LpH:5.8~6.0;(3)Differentiationmedium:MS+BA2mg/L+NAAImg/L+CoCl25mg/L+ZTO.5mg/L+Sucrose309门L+Inositol100rag/L+Agar6.59/LpH:5.8~6.O;(4)therootingmedium:1/2MS+IAA0,5mg几+NAA0.5mg/L+VCO.1%+Sucrose3
8、09L+Agar6.Sg/LpS:5.8~6.0.SomeregeneratedplantswereobtainedbyselectingforKanamycinresistanceof300mg/L.GenomicPC&Southernblott
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