小黑杨ftlike基因的克隆、原核表达与植物超表达、rnai表达载体的构建

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1、AbstractAftervegetativegrowthinjuvenility，treesusuallyturnintotheperiodofreproductivegrowthofanthesisthroughtheresponsetoaseriesofintemalandexternalfactors(such舔vernalization，etc)，andvegetativereproductionoftreeswillslowdownandstagnate．Inplant，FTgene，encodingaMADS-boxproteinwh

2、ichisactivatedbyCO，SOCl，AGL24andothergeneandsuppressedbyFLC,canpromotebyactivatingthefloralmeristemspecificgenesAPlandLFY．SointhecaseoftheexpressionlevelofFTgenecoulddeclineinpopulus，thefloweringofthepopuluswillbedelayedandmaintaininvegetativegrowthperiod(juvenilephase)togreat

3、lyincreasethetimberproduction．Onthecontrary，ifFTgenecouldoverexpressinpopulus，theanthesiswillbepromotedtoacceleratetheprocessandshortenthecycleofbreedingofpopulus．TotalRNAhasbeenextractedformtheleavesofPopulusXxiaohei，andthena535bpeDNAsequencehaSbeenclonedandcomparedthehomolog

4、ybetweenAtFTgeneofArabidopsisthaliana峨瓶a70％similarity．WenameditPnFTl．WeobtainedthePnFTI-pET-52brecombinantvectorbytheinsertionofPnFTlintotheMCSonpET-52bprokaryoticexpressionvector．BecauseofthepredictiveanalysisoftheE．colirarecodonsofPnFTlgeneshowing24．5％probability,weselectedt

5、heRosettaTMofE．coliasexpressstrains．AftertheinductionofTheRosettaTMofE．coliexpressstrainscarryingPnFTl一pET-52brecombinantvectorbyIPTC写wehavefoundathe28KDPnFTlrecombinantinclusionproteinbytheSDS—PAGEdetectionanalysisoftheextractionproductsoftotalproteinofthallus．Thepurification

6、PnFTIrecombinantproteinhasrevertedtothesolvableandactivatedconditionthroughtheextractionofinclusion,thepurificationofelectroelution,dialysisandprecipitationbyacetone．TheproteinsamplewithalOug／ulconcentrationand90％puritydidn’tcontainthematerialswhichalenotbeneficialtotheprepara

7、tionofthepolyclonalantibodysuchaSSDS，urea,CBB(Coomassiebrilliantblue)andSOon．ThePnFTlproteinhasabsolutelyreachingthestandardofthesampleforthepreparationofthepolyclonalantibodyandbeensenttoAbmartcompanytofinishthepreparation．WehavetransformedtheplantoverexpressionandRNAiexpress

8、ionvectorsofPnFTlgenebyIn—fusionandtraditionalmethodtoresearc