甘蔗C-,4-磷酸烯醇式丙酮酸羧化酶基因植物表達(dá)載體構(gòu)建及轉(zhuǎn)化大豆研究.pdf

1、福建農(nóng)林大學(xué)博士學(xué)位論文甘蔗C磷酸烯醇式丙酮酸羧化酶基因植物表達(dá)載體構(gòu)建及轉(zhuǎn)化大豆研究姓名：楊榮仲申請學(xué)位級別：博士專業(yè)：作物栽培學(xué)與耕作學(xué)指導(dǎo)教師：陳如凱2002.4.1‘、ConstructionofC4PhosphoenolpyruvateCarboxylaseGenefromSugarcaneandGeneticTransformationofSoybean(Glycinemax(L)Merrill)AbstractThreep

2、lantexpressionvectorswereconstructedbyinsertingtheC4phosphoenolpyruvatecarboxylasegenewhichincludesallintrons；partial5’flankingandpartial3’endingsequencewith68Kbfromsugarcane，intopBll21gettingpBIpepcwithaonecopyCaMV35Sas

3、promoterandnptllasselectablemarkergene；intopCAMBIAl301geeingpLApepcwithnopromoterandhygasselectablemarkergene，intopCAMBIAl301geeingpCBIpepcwithatwocopyCaMV35SaspromoterandhygasselectablemarkergeneThen，theywereintroducedi

4、ntoAgrobacteriumtumefaciensstrainLBA4404viathefreezemeltingTheprotocoloftransformationforsoybean(Glycinemox陋JMerrill)cotyledonarynodehasbeenoptimizedSoybean’scotyledonarynodewhichwasculturedwith10mg／LBAandlowtemperaturef

5、orafewdayswastransformedbySonicationAssistedAgrobacteriumtumefaciensmediatedmethodThetransformedcotyledonarynodeandshootswerescreenedundertheselectivepressureofKanorHygThetransformedplantsofpBIpepcfromAgrobacteriumtumefa

6、ciens—mediatedandpollentubeweredetectedbyPCRanddotSouthernboltingInordertocomparetheexpressionefficiencyofdifferenttypesofpromotersforsugarcaneC4phosphoenolpyruvateearboxylasegeneintransgenicC3plants，Threeplantexpression

7、vectorsweretransformedintotobaccobyAgrobacteriumtumefaciensTheresultswereasfollows：1ThedirectionofsugarcaneC4phosphoenotpyruvatecarboxylasegenewasidentifiedbypartial5’flankingand3’endingsequencethroughtheBlastnanalysis2T

8、hreeplantexpressionvectorspLApepc，pBIpepcandpCBIpepcwereconstructedbyinsertedtheC4phosphoenolpyruvatecarboxylasegenefromsugarcaneintopBll21andpCAMBIA1301TheselectablemarkergeneofpBlpepcwasnot／／andtheselectablemarkergeneo