定位于叶绿体的苏云金杆菌Bt杀虫蛋白表达载体的构建和转化水稻的研究

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1、中山大学硕士学位论文定位于叶绿体的苏云金杆菌（Bt）杀虫蛋白表达载体的构建和转化水稻的研究姓名：李华鹏申请学位级别：硕士专业：遗传学指导教师：许新萍2003.6.10至兰璺主些查兰堡主笙奎：塞垡三生堡竺竺莶墨垒堑堕!里!墨皇里旦塞竺墼竺堕塑塞塑簦些查塑竺堡塑AbstractBacillusthuringiensis(Bt)insecticidalcrystalprotein(ICP)geneistheontyICPgeneusedinthecommercialtransgeniccrops．AccordingtOthe“High·dose／re

2、fuge”tactic，whichprovedtobeusefulindelayingthedevelopmentofinsectresistancetotheICP,theinsufficientexpressionofBtICPinthetransgeniccropscouldresultinthesurvivaloftheinsectscarryingrecessiveresistantgene．ThelowexpressionofforeigngenesintransgenicplantsisalsoacommonproblemtObe

3、solvedinplantgeneticengineering．Toincreasetheexpressionofforeigngenesintransgenicplants，targetingtheforeignproteinstOthechloroplastisapracticalapproach．Twochloroplasttransitpeptide(CTP)genesequences，derivedfromriceandArabidopsisthalianarespectively，wereclonedbyPCRmethod．Andt

4、henbasedonthebinaryvectorpCAMBIA一1300whichisawidelyusedinplanttransformation，threeplantexpressionvectors，p13AOBcontainingCrylAc3genewithriceCTP,p13AABcontainingCrylAc3genewithArabidops&CTPandpI3ABcontainingCrylAc3genewithoutCTP,wereconstructed，TheexpressionofCrylAc3geneinall

5、threevectorsisdrivenbytheActlpromoterfromrice，whichcontaininganintronthatcanincreasetheexpressionfor10一foldormore．ThecodonsofCrylAc3geneinthesevectorsweremodifiedtOoptimizeitstranslationintransgenicplants．ThesethreevectorswereusedtOtransformtworicecultivars”Taipei309”and”Shi

6、shoubaimao”viaAgrobacterium—mediatedtransformationandpaniclebombardmentmethod．Eighty—sevenindependenttransgenicricelineswereobtainedonthemediumcontainingthe50mg／Lhygromycin，．SouthernblotanalysisofsomeofthetransgenicriceindicatedtheCrylAc3genehasintegratedintothericegenome．Th

7、eresultsofPCRdetectionofCrylAc3geneandhptgeneinmosttransgenicplantsshowedthat77．4％ofplantsharboringCrylAc3geneand96．9％plantsharboringhptgene．PCRanalysisofthetransgenicplantsalsorevealedthat85．7％oftheplantsobtainedbyAgrobacterium．mediatedtransformationand61．1％oftheplantsobtai

8、nedbyparticlebombardmentmethodcontainedbothCrylAc3geneand舾fgene，indicatingt