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海洋微拟球藻microRNA测序鉴定及其转基因体系的建立 Introduction Marinemicroalgaearewidelydistributedinoceansandareimportantprimaryproducersinthemarineecosystem.Amongthem,Emilianiahuxleyi,acoccolithophore,isadominantspeciesthatcontributessignificantlytotheglobalcarboncycle.Inaddition,E.huxleyihasbeenconsideredapotentialcandidateforbiofuelsandbioremediationduetoitshighlipidandcarbohydratecontent. MicroRNAs(miRNAs)areaclassofsmallnon-codingRNAsthatplayimportantregulatoryrolesingeneexpression.However,littleisknownaboutthemiRNA-mediatedpost-transcriptionalregulationofgeneexpressioninmarinemicroalgae,especiallyinE.huxleyi. Inthisstudy,weaimedtoidentifyandcharacterizemiRNAsinE.huxleyiusinghigh-throughputsequencingtechnology.Furthermore,weestablishedatransgenicsystembasedonmiRNA-mediatedgenesilencingtoinvestigateitspotentialapplicationinmarinemicroalgalbiotechnology. MaterialsandMethods SamplePreparationandRNAExtraction E.huxleyistrainCCMP1516wasculturedinartificialseawaterat20°Cunderalightintensityof60μmolphotonsm-2s-1witha16h:8hlight/darkcycle.TotalRNAwasextractedusingTRIzolreagent(Invitrogen)followingthemanufacturer'sinstructions.RNAqualityandintegritywereassessedusinganAgilent2100Bioanalyzer. SmallRNALibraryConstructionandSequencing Approximately1μgoftotalRNAfromE.huxleyiwasusedtopreparethesmallRNAlibraryusingtheNEBNext®MultiplexSmallRNALibraryPrepSetforIllumina®followingthemanufacturer'sprotocol.TheconstructedlibrarywassequencedonanIlluminaHiSeq2500platform. DataAnalysis Therawdatafromthesequencingwasprocessedviaqualitycontrol,adaptersremoval,andrRNAfilteringusingtheCutadaptandFastQCprograms.ThecleanreadswerealignedtotheE.huxleyigenomeusingtheBowtieprogram.ThemiRNAswereidentifiedusingthemiRDeep2software.ThetargetgenesofmiRNAswerepredictedusingthepsRNATargetserver. miRNA-mediatedGeneSilencing ToestablishamiRNA-mediatedgenesilencingsystem,wedesignedartificialmiRNAs(amiRNAs)targetingthetriosephosphateisomerase(TPI)geneofE.huxleyi.TheamiRNAswereclonedintoapCAMBIA2300vectorunderthecontroloftheU6